Nematode fatty acid desaturase-like sequences

ABSTRACT

Nucleic acid molecules from nematodes encoding fatty acid desaturase polypeptides are described. Fatty acid desaturase-like polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of fatty acid desaturase-like nucleotides and polypeptides. Also described are screening methods for identifying inhibitors and/or activators of fatty acid desaturase-like polypeptides, as well as methods for antibody production.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.12/061,072, filed Apr. 2, 2008, which is a divisional of U.S.application Ser. No. 11/060,008, filed Feb. 17, 2005, now abandonedwhich is a divisional of U.S. application Ser. No. 10/243,468, filedSep. 13, 2002, now U.S. Pat. No. 6,875,595 which claims priority to U.S.provisional application Ser. No. 60/322,003, filed Sep. 13, 2001, all ofwhich are herein incorporated by reference.

BACKGROUND

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingthe roots, developing flower buds, leaves, and stems. Plant parasitesare classified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the United States (Carter (2001) CaliforniaAgriculture, 55(3):2). It is expected that strawberry and othercommodity crop industries will be significantly impacted if a suitablereplacement for methyl bromide is not found. Presently there are a verysmall array of chemicals available to control nematodes and they arefrequently inadequate, unsuitable, or too costly for some crops or soils(Becker (1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat.Nos. 6,048,714). The few available broad-spectrum nematicides such asTelone (a mixture of 1,3-dichloropropene and chloropicrin) havesignificant restrictions on their use because of toxicological concerns(Carter (2001) California Agriculture, Vol. 55(3):12-18).

Fatty acids are a class of natural compounds that have been investigatedas alternatives to the toxic, non-specific organophosphate, carbamateand fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this general mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), as bactericides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides(e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).

The phytotoxicity of fatty acids has been a major constraint on theirgeneral use in agricultural applications (U.S. Pat. No. 5,093,124) andthe mitigation of these undesirable effects while preserving pesticidalactivity is a major area of research. The esterilication of fatty acidscan significantly decrease their phytotoxicity (U.S. Pat. Nos.5,674,897; 5,698,592; 6,124,359). Such modifications can however lead todramatic loss of nematicidal activity as is seen for linoleic, linolenicand oleic acid (Stadler et al. (1994) Planta Medica 60(2): 128-132) andit may be impossible to completely decouple the phytotoxicity andnematicidal activity of pesticidal fatty acids because of theirnon-specific mode of action. Perhaps not surprisingly, the nematicidalfatty acid pelargonic acid methyl ester (U.S. Pat. Nos. 5,674,897;5,698,592; 6,124,359) shows a relatively small “therapeutic window”between the onset of pesticidal activity and the observation ofsignificant phytotoxicity (Davis et al. (1997) J Nematol29(4S):677-684). This is the expected result if both the phytotoxicityand the nematicidial activity derive from the non-specific disruption ofplasma membrane integrity. Similarly the rapid onset of pesticidalactivity seen with many nematicidal fatty acids at therapeuticconcentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggestsa non-specific mechanism of action, possibly related to the disruptionof membranes, action potentials and neuronal activity.

Ricinoleic acid, the major component of castor oil, provides anotherexample of the unexpected effects esterification can have on fatty acidactivity. Ricinoleic acid has been shown to have an inhibitory effect onwater and electrolyte absorption using everted hamster jejunal and ilealsegments (Gaginella et al. (1975) J Pharmacol Exp Ther 195(2):355-61)and to be cytotoxic to isolated intestinal epithelial cells (Gaginellaet al. (1977) J Pharmacol Exp Ther 201(1):259-66). These features arelikely the source of the laxative properties of castor oil, which isgiven as a purgative in humans and livestock, e.g., as a dewormingagent. In contrast, the methyl ester of ricinoleic acid is ineffectiveat suppressing water absorption in the hamster model (Gaginella et al.(1975) J Pharmacol Exp Ther 195(2):355-61).

The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that inprinciple provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently, despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta-toxins must be ingested to affect their target organ, the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics.155(4): 1693-1699). Consequently they are not anticipated to beeffective against the dispersal, non-feeding, juvenile stages of plantparasitic nematodes in the field. These juvenile stages only commencefeeding when a susceptible host has been infected. Thus, Bt delta-toxinsnematicides may need to penetrate the cuticle in order to be effective.In addition, soil mobility of a relatively large 65-130 kDa protein—thesize of typical Bt delta-toxins—is expected to be poor and delivery inplanta is likely to be constrained by the exclusion of large particlesby the feeding tube of certain plant parasitic nematodes such asHeterodera (Atkinson et al. (1998) Engineering resistance toplant-parasitic nematodes. In: The Physiology and Biochemistry ofFree-Living and Plant-parasitic Nematodes (eds R. N. Perry & D. J.Wright), CAB International 1998).

Many plant species are known to be highly resistant to nematodes. Thebest documented of these include marigolds (Tageles spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). The active principle(s) for thisnematicidal activity has not been discovered in all of these examplesand no plant-derived products are sold commercially for control ofnematodes. In the case of the Asteraceae, the photodynamic compoundalpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots. Castor beans are plowed under as a green manurebefore a seed crop is set. However, a significant drawback of the castorplant is that the seed contains toxic compounds (such as ricin) that cankill humans, pets, and livestock and is also highly allergenic.

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

Nematode parasites of vertebrates (e.g., humans, livestock and companionanimals) include gut roundworms, hookworms, pinworms, whipworms, andfilarial worms. They can be transmitted in a variety of ways, includingby water contamination, skin penetration, biting insects, or byingestion of contaminated food.

In domesticated animals, nematode control or “de-worming” is essentialto the economic viability of livestock producers and is a necessary partof veterinary care of companion animals. Parasitic nematodes causemortality in animals (e.g., heartworm in dogs and cats) and morbidity asa result of the parasites' inhibiting the ability of the infected animalto absorb nutrients. Parasite-induced nutrient deficiency results indiseased livestock and companion animals (i.e., pets), as well as instunted growth. For instance, in cattle and dairy herds, a singleuntreated infection with the brown stomach worm can permanently stunt ananimal's ability to effectively convert feed into muscle mass or milk.

Two factors contribute to the need for novel anthelmintics and vaccinesfor control of parasitic nematodes of animals. First, some of the moreprevalent species of parasitic nematodes of livestock are buildingresistance to the anthelmintic drugs available currently, meaning thatthese products will eventually lose their efficacy. These developmentsare not surprising because few effective anthelmintic drugs areavailable and most have been used continuously. Presently a number ofparasitic species has developed resistance to most of the anthelmintics(Geents et al. (1997) Parasitology Today 13:149-151; Prichard (1994)Veterinary Parasitology 54:259-268). The fact that many of theanthelmintic drugs have similar modes of action complicates matters, asthe loss of sensitivity of the parasite to one drug is often accompaniedby side resistance—that is, resistance to other drugs in the same class(Sangster & Gill (1999) Parasitology Today Vol. 15(4): 141-146).Secondly, there are some issues with toxicity for the major compoundscurrently available.

Human infections by nematodes result in significant mortality andmorbidity, especially in tropical regions of Africa, Asia, and theAmericas. The World Health Organization estimates 2.9 billion people areinfected with parasitic nematodes. While mortality is rare in proportionto total infections (180,000 deaths annually), morbidity is tremendousand rivals tuberculosis and malaria in disability adjusted life yearmeasurements. Examples of human parasitic nematodes include hookworm,filarial worms, and pinworms. Hookworm is the major cause of anemia inmillions of children, resulting in growth retardation and impairedcognitive development. Filarial worm species invade the lymphatics,resulting in permanently swollen and deformed limbs (elephantiasis) andinvade the eyes causing African Riverblindness. Ascaris lumbricoides,the large gut roundworm infects more than one billion people worldwideand causes malnutrition and obstructive bowl disease. In developedcountries, pinworms are common and often transmitted through children indaycare.

Even in asymptomatic parasitic infections, nematodes can still deprivethe host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

While public health measures have nearly eliminated one tropicalnematode (the water-borne Guinea worm), cases of other worm infectionshave actually increased in recent decades. In these cases, drugintervention provided through foreign donations or purchased by thosewho can afford it remains the major means of control. Because of thehigh rates of reinfection after drug therapy, vaccines remain the besthope for worm control in humans. There are currently no vaccinesavailable.

Until safe and effective vaccines are discovered to prevent parasiticnematode infections, anthelmintic drugs will continue to be used tocontrol and treat nematode parasitic infections in both humans anddomestic animals. Finding effective compounds against parasiticnematodes has been complicated by the fact that the parasites have notbeen amenable to culturing in the laboratory. Parasitic nematodes areoften obligate parasites (i.e., they can only survive in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

C. elegans is a small free-living bacteriovorous nematode that for manyyears has served as an important model system for multicellular animals(Burglin (1998) Int. J. Parasitol., 28(3): 395-411). The genome of C.elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282: 2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30).

Although C. elegans serves as a good model system for vertebrates, it isan even better model for study of parasitic nematodes, as C. elegans andother nematodes share unique biological processes not found invertebrates. For example, unlike vertebrates, nematodes produce and usechitin, have gap junctions comprised of innexin rather than connexin andcontain glutamate-gated chloride channels rather than glycine-gatedchloride channels (Bargmann (1998) Science 282: 2028-33). The latterproperty is of particular relevance given that the avermectin class ofdrugs is thought to act at glutamate-gated chloride receptors and ishighly selective for invertebrates (Martin (1997) Vet. J. 154:11-34).

A subset of the genes involved in nematode specific processes will beconserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene can destroy expressedsequences corresponding to the selected gene thus reducing expression ofthe corresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

SUMMARY

The invention features nucleic acid molecules encoding Meloidogyneincognita, Heterodera glycines, Diroilaria immitis, Strongyloidesstercoralis and Rhabditella axei fatty acid desaturases and othernematode fatty acid desaturase-like proteins. M. incognita is a RootKnot Nematode that causes substantial damage to several crops, includingcotton, tobacco, pepper, and tomato. H. glycines, referred to as SoybeanCyst Nematode, is a major pest of soybean. D. immitis (dog heartworm)and S. stercoralis (human threadworm) are mammalian parasites. R. axeiis a free-living nematode that serves as a model for parasiticnematodes. In part, the fatty acid desaturase-like nucleic acids andpolypeptides of the invention allow for the identification of a nematodespecies, and for the identification of compounds that bind to or alterthe activity of fatty acid desaturase-like polypeptides. Such compoundsmay provide a means for combating diseases and infestations caused bynematodes, particularly those caused by M. incognita (e.g., in tobacco,cotton, pepper, or tomato plants), H. glycines (e.g., in soybeans), D.immitis (e.g., in dogs) and S. stercoralis (e.g., in humans).

The invention is based, in part, on the identification of cDNAs encodingM. incognita fatty acid desaturases (SEQ ID NO: 1, SEQ ID NO: 2 and SEQID NO: 3). These 1194, 1242 and 1260 nucleotide cDNAs have 1191, 1239and 1161 nucleotide open reading frames encoding 397, 413 and 387 aminoacid polypeptides (SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10)respectively.

The invention is also based, in part, on the identification of a cDNAencoding H. glycines fatty acid desaturase (SEQ ID NO: 4). This 1488nucleotide cDNA has a 1167 nucleotide open reading frame encoding a 389amino acid polypeptide (SEQ ID NO: 11).

The invention is also based, in part, on the identification of a partialcDNA fragment encoding D. immitis fatty acid desaturase (SEQ ID NO: 5).This 1068 nucleotide cDNA has a 867 nucleotide open reading frameencoding a 289 amino acid polypeptide (SEQ ID NO: 12).

The invention is also based, in part, on the identification of a cDNAencoding S. stercoralis fatty acid desaturase (SEQ ID NO: 6). This 1221nucleotide cDNA has a 1104 nucleotide open reading frame encoding a 368amino acid polypeptide (SEQ ID NO: 13).

The invention is also based, in part, on the identification of a cDNAencoding R. axei fatty acid desaturase (SEQ ID NO: 7). This 1233nucleotide cDNA has a 1122 nucleotide open reading frame encoding a 374amino acid polypeptide (SEQ ID NO: 14).

In one aspect, the invention features novel nematode fatty aciddesaturase-like polypeptides. Such polypeptides include purifiedpolypeptides having the amino acid sequences set forth in SEQ ID NO: 8,9, 10, 11, 12, 13 and 14. Also included are polypeptides having an aminoacid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, or98% identical to SEQ ID NO: 8, 9, 10, 11, 12, 13 or 14. The purifiedpolypeptides can be encoded by a nematode gene, e.g., a nematode geneother than a C. elegans gene. For example, the purified polypeptide hasa sequence other than SEQ ID NO: 32 (C. elegans fatty acid desaturase).The purified polypeptides can further include a heterologous amino acidsequence, e.g., an amino-terminal or carboxy-terminal sequence.

Also featured are purified polypeptide fragments of the aforementionedfatty acid desaturase-like polypeptides, e.g., a fragment of at leastabout 20, 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 325, 350, 375,395, 400 amino acids. Non-limiting examples of such fragments include:fragments from about amino acid 1 to 50, 1 to 75, 1 to 92, 1 to 96, 1 to100, 1 to 125, 1 to 399, 51 to 100, 92 to 150, 96 to 150, 92 to 400, 200to 300, and 1 to 380 of SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14. Thepolypeptide or fragment thereof can be modified, e.g., processed,truncated, modified (e.g. by glycosylation, phosphorylation,acetylation, myristylation, prenylation, palmitoylation, amidation,addition of glycerophosphatidyl inositol), or any combination of theabove. These various polypeptide fragments can be used for a variety ofpurposes, including for the eliciting of antibodies directed against afatty acid desaturase-like polypeptide.

In another aspect, the invention features novel isolated nucleic acidmolecules encoding nematode fatty acid desaturase-like polypeptides.Such isolated nucleic acid molecules include nucleic acids comprising,consisting of or consisting essentially of the nucleotide sequence setforth in SEQ ID NO: 1, 2, 3, 4, 5, 6 and 7. Also included are isolatednucleic acid molecules having the same sequence as or encoding the samepolypeptide as a nematode fatty acid desaturase-like gene (other than C.elegans fatty acid desaturase-like genes).

Also featured are: 1) isolated nucleic acid molecules having a strandthat hybridizes under low stringency conditions to a single strandedprobe of the sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 or theircomplements and, optionally, encodes polypeptides of between 375 and 425amino acids and preferably have Δ12 fatty acid desaturase activity; 2)isolated nucleic acid molecules having a strand that hybridizes underhigh stringency conditions to a single stranded probe of the sequence ofSEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 or their complements and,optionally, encodes polypeptides of between 375 and 425 amino acids andpreferably have Δ12 fatty acid desaturase activity; 3) isolated nucleicacid fragments of a fatty acid desaturase-like nucleic acid molecule,e.g., a fragment of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 that is about 230,435, 450, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1450 or morenucleotides in length or ranges between such lengths; and 4)oligonucleotides that are complementary to a fatty acid desaturase-likenucleic acid molecule or a fatty acid desaturase-like nucleic acidcomplement, e.g., an oligonucleotide of about 10, 15, 18, 20, 22, 24,28, 30, 35, 40, 50, 60, 70, 80, or more nucleotides in length. Exemplaryoligonucleotides are oligonucleotides which anneal to a site locatedbetween nucleotides about 1 to 24, 1 to 48, 1 to 60, 1 to 120, 24 to 48,24 to 60, 49 to 60, 61 to 180, 145 to 165, 165 to 185, 1260 to 1280,1281 to 1300, 1301 to 1320, 1321 to 1340, 1341 to 1360, 1361 to 1380,1381 to 1400, 1401 to 1420, 1421 to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6or 7. Such nucleic acid fragments are useful for detecting the presenceof fatty acid desaturase-like mRNA. Nucleic acid fragments include thefollowing non-limiting examples: nucleotides about 1 to 200, 100 to 300,200 to 400, 300 to 500, 400 to 600, 500 to 700, 600 to 800, 700 to 900,800 to 1000, 900 to 1100, 1000 to 1200, 1100 to 1300, 1200 to 1400, 1300to 1456 of SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7. Also within the inventionare nucleic acid molecules that hybridize under stringent conditions tonucleic acid molecules comprising SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7 andcomprise 3,000, 2,000, 1,000 or fewer nucleotides and preferably encodea polypeptide having Δ12 fatty acid desaturase activity and/or have asequence corresponding to that of a naturally occurring nematode. Theisolated nucleic acid can further include a heterologous promoteroperably linked to the fatty acid desaturase-like nucleic acid molecule.

A molecule featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the moleculecan be from a species of the class Rhabditida, particularly a speciesother than C. elegans.

In another aspect, the invention features a vector, e.g., a vectorcontaining an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoter.The regulatory elements can be operably linked to the fatty aciddesaturase-like nucleic acid molecules in order to express a fatty aciddesaturase-like nucleic acid molecule. In yet another aspect, theinvention features a transgenic cell or transgenic organism having inits genome a transgene containing an aforementioned fatty aciddesaturase-like nucleic acid molecule and a heterologous nucleic acid,e.g., a heterologous promoter.

In still another aspect, the invention features an antibody, e.g., anantibody, antibody fragment, or derivative thereof that bindsspecifically to an aforementioned polypeptide. Such antibodies can bepolyclonal or monoclonal antibodies. The antibodies can be modified,e.g., humanized, rearranged as a single-chain, or CDR-grafted. Theantibodies may be directed against a fragment, a peptide, or adiscontinuous epitope from a fatty acid desaturase-like polypeptide.

In another aspect, the invention features a method of screening for acompound that binds to a nematode fatty acid desaturase-likepolypeptide, e.g., an aforementioned polypeptide. The method includesproviding the nematode polypeptide; contacting a test compound to thepolypeptide; and detecting binding of the test compound to the nematodepolypeptide. In one embodiment, the method further includes contactingthe test compound to a mammalian or plant fatty acid desaturase-likepolypeptide; and detecting binding of the test compound to the mammalianor plant fatty acid desaturase-like polypeptide. A test compound thatbinds the nematode fatty acid desaturase-like polypeptide with at least2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold affinity greaterrelative to its affinity for the mammalian (e.g., a human) or plantfatty acid desaturase-like polypeptide can be identified.

Alternatively, the compounds can bind to the mammalian or plant fattyacid desaturase with affinity similar to that of the nematode fatty aciddesaturase, but not be significantly detrimental to a plant and/oranimal.

The invention also features methods for identifying compounds that alterthe activity of a nematode fatty acid desaturase-like polypeptide. Themethod includes contacting the test compound to the nematode fatty aciddesaturase-like polypeptide and detecting a fatty acid desaturase-likeactivity. A decrease in the level of fatty acid desaturase-like activityof the polypeptide relative to the level of fatty acid desaturase-likeactivity of the polypeptide in the absence of the test compound is anindication that the test compound is an inhibitor of the fatty aciddesaturase-like activity. In still another embodiment, the methodfurther includes contacting a nematode fatty acid desaturase polypeptidewith a test compound such as an allosteric inhibitor, a substrateanalog, other analogs, or other types of inhibitors that prevent bindingof the fatty acid desaturase-like polypeptide to other molecules orproteins (“fatty acid desaturase-like polypeptide binding partners”). Achange in activity or fatty acid desaturase-like polypeptide binding ofproteins normally bound by the fatty acid desaturase is an indicationthat the test compound is an inhibitor of the fatty acid desaturase-likeactivity or is an inhibitor of the interaction of the fatty aciddesaturase-like polypeptide with one of its binding partners. Suchinhibitory compounds are potentially selective agents for reducing theviability, growth, development or reproduction of a nematode expressinga fatty acid desaturase-like polypeptide, e.g., M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei. These methods can alsoinclude contacting the test compound with a plant or mammalian (e.g., ashuman) fatty acid desaturase-like polypeptide; and detecting a fattyacid desaturase-like activity of the plant or mammalian fatty aciddesaturase-like polypeptide. A compound that decreases nematode fattyacid desaturase activity to a greater extent than it decreases a plantor mammalian fatty acid desaturase-like polypeptide activity is acandidate selective inhibitor of nematode viability, growth orreproduction. A desirable compound can exhibit 2-fold, 5-fold, 10-fold,20-fold, 50-fold, 100-fold or greater selective activity against thenematode polypeptide. Any suitable assay can be used to measure fattyacid desaturase activity, including that of Banas et al. (Physiology,Biochemistry and Molecular Biology of Plant Lipids, Williams et al.,eds., Kluwer Academic, Dordrecht, Netherlands, 1996, pages 57-59).

Another featured method is a method of screening for a compound thatalters an activity of a fatty acid desaturase-like polypeptide or altersbinding or regulation of other polypeptides by fatty acid desaturase.Thus, the activity of the fatty acid desaturase can be measured directlyby monitoring the substrate or product of the fatty acid desaturase orindirectly by measuring the activity (e.g., monitoring the substrate orproduct) of an enzyme that acts downstream of fatty acid desaturase.Thus, the methods of the invention include providing a fatty aciddesaturase polypeptide; contacting a test compound to the polypeptide;and detecting a fatty acid desaturase-like activity of the polypeptideor the activity of polypeptides bound or regulated by the fatty aciddesaturase, wherein a change in activity of the fatty aciddesaturase-like polypeptide or other downstream polypeptides relative tothe fatty acid desaturase-like activity of the polypeptide or downstreampolypeptides in the absence of the test compound is an indication thatthe test compound alters the activity of the polypeptide(s). Similarly,the method includes providing the polypeptide; contacting a testcompound to the polypeptide; and detecting a fatty acid desaturase-likeactivity or the resulting unsaturated fatty acid products, wherein achange in activity of fatty acid desaturase-like polypeptides or theresulting unsaturated fatty acid products relative to the fatty aciddesaturase-like activity of the polypeptide or the level of fatty acidproducts in the absence of the test compound is an indication that thetest compound alters the activity of the polypeptide(s).

The methods of the invention can include contacting the test compound toa plant or mammalian (e.g., a human) fatty acid desaturase-likepolypeptide and measuring the fatty acid desaturase-like activity of theplant or mammalian fatty acid desaturase-like polypeptide or otherpolypeptides affected or regulated by the fatty acid desaturase or theresulting unsaturated fatty acid products. A test compound that altersthe activity of the nematode fatty acid desaturase-like polypeptide (orthe level of fatty acid products) at a given concentration and that doesnot substantially alter the activity of the plant or mammalian fattyacid desaturase-like polypeptide, downstream polypeptides, or level offatty acid products at the same given concentration can be identified.Thus, the methods of the invention can be used to identify candidatecompounds that are relatively selective for one or more nematode fattyacid desaturase-like polypeptides relative to one or more mammalian andor plant fatty acid desaturase-like polypeptides. An additional methodincludes screening for both binding to a fatty acid desaturase-likepolypeptide and for an alteration in the activity of a fatty aciddesaturase-like polypeptide.

The methods of the invention include the identification of compoundsthat inhibit both nematode and plant fatty acid desaturase-likepolypeptides. Such compounds can be useful for treatment of preventionof nematode infection of plants because plants are often notsignificantly impaired by inhibition of the activity of a fatty aciddesaturase-like polypeptide. Moreover, such inhibitors can beadministered to a mammal for treatment or prevention of infection by anematode.

Yet another featured method is a method of screening for a compound thatalters the viability or fitness of a transgenic cell or organism (e.g.,a nematode). The transgenic cell or organism has a transgene thatexpresses a fatty acid desaturase-like polypeptide. The method includescontacting a test compound to the transgenic cell or organism anddetecting changes in the viability or fitness of the transgenic cell ororganism. This alteration in viability or fitness can be measuredrelative to an otherwise identical cell or organism that does not harborthe transgene.

The invention also features compounds that are relatively selectiveinhibitors of one or more nematode fatty acid desaturase-likepolypeptides relative to one or more plant or animal fatty aciddesaturase-like polypeptides. The compounds can have a K_(i) for anematode fatty acid desaturase that is 10-fold, 100-fold, 1,000-fold ormore lower than for a plant or animal fatty acid desaturase-likepolypeptides, e.g., a host plant or host animal of the nematode. Theinvention further features relatively non-selective inhibitors as wellas completely non-selective inhibitors.

Also featured is a method of screening for a compound that alters theexpression of a nematode nucleic acid encoding a fatty aciddesaturase-like polypeptide or nucleic acid encoding a nematode fattyacid desaturase-like polypeptide, e.g., a nucleic acid encoding a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide. The method includes contacting a cell,e.g., a nematode cell, with a test compound and detecting expression ofa nematode nucleic acid encoding a fatty acid desaturase-likepolypeptide, e.g., by hybridization to a probe complementary to thenematode mRNA encoding an fatty acid desaturase-like polypeptide or bycontacting polypeptides isolated from the cell with a compound, e.g.,antibody that binds a fatty acid desaturase-like polypeptide.

In yet another aspect, the invention features a method of treating adisorder (e.g., an infection) caused by a nematode, e.g., M. incognita,H. glycines, D. immitis or S. stercoralis, in a subject, e.g., a hostplant or host animal. The method includes administering to the subjectan effective amount of an inhibitor of a fatty acid desaturase-likepolypeptide activity or an inhibitor of expression of a fatty aciddesaturase-like polypeptide. Non-limiting examples of such inhibitorsinclude: an antisense nucleic acid (or PNA) to a fatty aciddesaturase-like nucleic acid, an antibody to a fatty aciddesaturase-like polypeptide, an analog of a natural substrate of a fattyacid desaturase, a fatty acid, or a small molecule identified as a fattyacid desaturase-like polypeptide inhibitor, e.g., an inhibitoridentified by a method described herein.

In another aspect, methods for desaturating fatty acids to Δ12 fattyacids are provided. Such methods can include the steps of: (a) providinga cell that harbors a fatty acid desaturase polypeptide; and (b) growingthe cell under conditions in which the fatty acid desaturase polypeptidedesaturates a fatty acid to produce a corresponding Δ12 unsaturatedfatty acid.

In still another aspect, methods of inhibiting nematode (e.g., M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei) Δ12 fattyacid desaturase(s) are provided. Such methods can include the steps of:(a) providing a nematode that expresses a Δ12 fatty acid desaturase-likeenzyme; (b) contacting the nematode with fatty acid analogs or othercompounds that inhibit the enzyme. Also provided are methods of rescuingthe effect of the inhibitor. Such methods comprise the steps of: (a)inhibiting the enzyme and (b) providing Δ12 unsaturated fatty acidsexogenously.

In another aspect, methods of reducing the viability or fecundity orslowing the growth or development or inhibiting the infectivity of anematode using a fatty acid analog or inhibitor of a fatty aciddesaturase are provided. Such methods comprise the steps of (a)providing a nematode that expresses a Δ12 fatty acid desaturase; (b)contacting the nematode with specific inhibitory fatty acid analogs orinhibitors of a Δ12 fatty acid desaturase; (c) reducing the viability orfecundity of the nematode. Also provided are methods of rescuing theeffect of the fatty acid desaturase inhibitors or other inhibitors. Suchmethods can involve contacting the nematode with Δ12 unsaturated fattyacids exogenously.

In another aspect, methods of inhibiting a Δ12 fatty acid desaturaseusing RNA interference methods are provided. Such methods comprise thesteps of (a) providing a nematode that contains a Δ12 fatty aciddesaturase like gene; (b) contacting the nematode with double strandedRNA (dsRNA). Such methods can be used to reduce viability or fecundity,to slow growth or development, or to inhibit infectivity. In anotheraspect, methods of rescuing the effect of RNA interference by supplyingspecific Δ12 unsaturated fatty acids are provided.

The methods of the invention include a method for identifying aninhibitor of a fatty acid desaturase-like polypeptide, the methodcomprising: (a) providing a cell expressing a fatty acid desaturase-likepolypeptide; (b) contacting the cell with a test compound; (c) measuringthe fatty acid desaturase-like polypeptide activity of the cell, whereina change in fatty acid desaturase-like polypeptide activity of the cellrelative to the fatty acid desaturase-like polypeptide activity of thecell in the absence of the test compound is an indication that the testcompound alters the activity of the fatty acid desaturase-likepolypeptide.

The methods of the invention further include a method for identifying aninhibitor of a fatty acid desaturase-like polypeptide, the methodcomprising: (a) providing a nematode expressing a fatty aciddesaturase-like polypeptide; (b) contacting the nematode with a testcompound; (c) measuring the fatty acid desaturase-like polypeptideactivity of the nematode, wherein a change in fatty acid desaturase-likepolypeptide activity of the nematode relative to the fatty aciddesaturase-like polypeptide activity of the nematode in the absence ofthe test compound is an indication that the test compound alters theactivity of the fatty acid desaturase-like polypeptide.

Another method for identifying an inhibitor of a fatty aciddesaturase-like polypeptide comprises: (a) providing a cell expressing afatty acid desaturase-like polypeptide; (b) contacting the cell with atest compound; (c) measuring the viability of the cell in the presenceof the test compound; and (d) comparing the viability of the cell in thepresence of the test compound to the viability of the cell in thepresence of the test compound and a product of the fatty aciddesaturase-like polypeptide, wherein greater viability in the presenceof the test compound and the product compared to viability in thepresence of the test compound is an indication that the test compoundalters the activity of the fatty acid desaturase-like polypeptide. Theinvention features a method for identifying an inhibitor of a fatty aciddesaturase-like polypeptide, the method comprising: (a) providing anematode expressing a fatty acid desaturase-like polypeptide; (b)contacting the nematode with a test compound; (c) measuring theviability or the fecundity of the nematode in the presence of the testcompound; and (d) comparing the viability or fecundity of the nematodein the presence of the test compound to the viability or fecundity ofthe nematode in the presence of the test compound and a product of thefatty acid desaturase-like polypeptide, wherein greater viability orfecundity in the presence of the test compound and the product comparedto viability or fecundity in the presence of the test compound is anindication that the test compound alters the activity of the fatty aciddesaturase-like polypeptide. In preferred embodiments the product islinoleic acid.

The invention also features inhibitors identified by the screeningmethods disclosed herein.

The invention features a method for reducing the viability, growth, orfecundity of a nematode, the method comprising exposing the nematode toan agent that inhibits the activity of a fatty acid desaturase-likepolypeptide (e.g., a Δ12 fatty acid desaturase) and a method forprotecting a plant from a nematode infection, the method comprisingapplying to the plant or to seeds of the plant an inhibitor of anematode fatty acid desaturase-like polypeptide. The invention alsofeatures a method for protecting a mammal from a nematode infection, themethod comprising administering to the mammal an inhibitor of a nematodefatty acid desaturase-like polypeptide (e.g., a Δ12 fatty aciddesaturase). In preferred embodiments the inhibitor does notsignificantly inhibit the activity of a fatty acid desaturase-likepolypeptide expressed by the plant or at least does not do so to theextent that the growth of the plant is impaired.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary. Isolated nucleic acid molecules according to the presentinvention further include molecules produced synthetically, as well asany nucleic acids that have been altered chemically and/or that havemodified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides in the nucleic acid molecule,the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (1990)Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLASTN and BLASTX programs (version 2.0 and2.1) of Altschul et al. (1990). J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTX program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402.When utilizing BLAST and Gapped BLAST programs for the determination ofpercent identity of amino acid sequences or nucleotide sequences, thedefault parameters of the respective programs can be used. The programsare available on the Internet at: www.ncbi.nlm.nih.gov.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one or more subject polypeptides), which is partly orentirely heterologous, i.e., foreign, to the transgenic plant, animal,or cell into which it is introduced, or, is homologous to an endogenousgene of the transgenic plant, animal, or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe plant's genome in such a way as to alter the genome of the cell intowhich it is inserted (e.g., it is inserted at a location which differsfrom that of the natural gene or its insertion results in a knockout). Atransgene can include one or more transcriptional regulatory sequencesand other nucleic acid sequences, such as introns, that may be necessaryfor optimal expression of the selected nucleic acid, all operably linkedto the selected nucleic acid, and may include an enhancer sequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant includes a transgene. The transgenecan be introduced into the cell, directly or indirectly by introductioninto a precursor of the cell, by way of deliberate genetic manipulation,such as by T-DNA mediated transfer, electroporation, or protoplasttransformation. The transgene may be integrated within a chromosome, orit may be extrachromosomally replicating DNA.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such as aleaf, root, seed, or stem.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refer to conditions forhybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, an agent with “anthelmintic activity” is an agent, whichwhen tested, has measurable nematode-killing activity or results inreduced fertility or sterility in the nematodes such that fewer viableor no offspring result, or compromises the ability of the nematode toinfect or reproduce in its host, or interferes with the growth ordevelopment of a nematode. In the assay, the agent is combined withnematodes, e.g., in a well of microtiter dish having agar media or inthe soil containing the agent. Staged adult nematodes are placed on themedia. The time of survival, viability of offspring, and/or the movementof the nematodes are measured. An agent with “anthelminthic activity”can, for example, reduce the survival time of adult nematodes relativeto unexposed similarly staged adults, e.g., by about 20%, 40%, 60%, 80%,or more. In the alternative, an agent with “anthelminthic activity” mayalso cause the nematodes to cease replicating, regenerating, and/orproducing viable progeny, e.g., by about 20%, 40%, 60%, 80%, or more.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in molar excess over the antibody.

As used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, (e.g., anincrease or decrease in the ability of the polypeptide to bind orregulate other polypeptides or molecules) particularly a fatty aciddesaturase-like or fatty acid desaturase activity (e.g., the ability tointroduce a double bond at the Δ12 position of a fatty acid). The changecan be detected in a qualitative or quantitative observation. If aquantitative observation is made, and if a comprehensive analysis isperformed over a plurality of observations, one skilled in the art canapply routine statistical analysis to identify modulations where a levelis changed and where the statistical parameter, the p value, is, forexample, less than 0.05.

In part, the nematode fatty acid desaturase proteins and nucleic acidsdescribed herein are novel targets for anti-nematode vaccines,pesticides, and drugs. Inhibition of these molecules can provide meansof inhibiting nematode metabolism, growth, viability, fecundity,development, infectivity and/or the nematode life-cycle.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 depicts the cDNA sequence of a M. incognita fatty acid desaturase(SEQ ID NO: 1), its corresponding encoded amino acid sequence (SEQ IDNO: 8).

FIG. 2 depicts the cDNA sequence of a second M. incognita fatty aciddesaturase (SEQ ID NO: 2), its corresponding encoded amino acid sequence(SEQ ID NO: 9).

FIG. 3 depicts the cDNA sequence of a third M. incognita fatty aciddesaturase (SEQ ID NO: 3), its corresponding encoded amino acid sequence(SEQ ID NO: 10).

FIG. 4 depicts the cDNA sequence of a II. glycines fatty acid desaturase(SEQ ID NO: 4), its corresponding encoded amino acid sequence (SEQ IDNO: 11).

FIG. 5 depicts the partial cDNA sequence of a D. immitis fatty aciddesaturase (SEQ ID NO: 5), its corresponding encoded amino acid sequence(SEQ ID NO: 12).

FIG. 6 depicts the cDNA sequence of a S. stercoralis fatty aciddesaturase (SEQ ID NO: 6), its corresponding encoded amino acid sequence(SEQ ID NO: 13).

FIG. 7 depicts the cDNA sequence of a R. axei fatty acid desaturase (SEQID NO: 7), its corresponding encoded amino acid sequence (SEQ ID NO:14).

FIG. 8 depicts an alignment of the sequences of M. incognita, H.glycines, D. immitis, S. stercoralis and R. axei fatty aciddesaturase-like polypeptides (SEQ ID NO: 8, 9, 10, 11, 12, 13 and 14 anda C. elegans Δ12 fatty acid desaturase polypeptide (SEQ ID NO: 32 (lowersequence)).

FIG. 9 is a photograph of C. elegans grown on oleic acid.

FIG. 10 is a photograph of C. elegans grown on linolecic acid.

FIG. 11 is a photograph of C. elegans grown on ricinoleic acid.

FIG. 12 is a photograph of C. elegans grown on vernolic acid.

DETAILED DESCRIPTION

Described below is the identification of M. incognita, H. glycines, D.immitis, S. stercoralis and R. axei fatty acid desaturase cDNAs (SEQ IDNO: 1, 2, 3, 4, 5, 6 and 7) and the polypeptides they encode (SEQ ID NO:8, 9, 10, 11, 12, 13 and 14). The fatty acid desaturases are Δ12 fattyacid desaturases. Also described below are experiments demonstratingthat the fatty acid desaturase is essential for nematode viability. Alsodescribed below are inhibitors of the fatty acid desaturase. Certainsequence information for the fatty acid desaturase genes describedherein is summarized in Table 1, below.

TABLE 1 cDNAs Identified Species cDNA Polypeptide Figure M. incognitaSEQ ID NO: 1 SEQ ID NO: 8 FIG. 1 M. incognita SEQ ID NO: 2 SEQ ID NO: 9FIG. 2 M. incognita SEQ ID NO: 3 SEQ ID NO: 10 FIG. 3 H. glycines SEQ IDNO: 4 SEQ ID NO: 11 FIG. 4 D. immitis SEQ ID NO: 5 SEQ ID NO: 12 FIG. 5S. stercoralis SEQ ID NO: 6 SEQ ID NO: 13 FIG. 6 R. axei SEQ ID NO: 7SEQ ID NO: 14 FIG. 7

Unsaturated fatty acids are essential to the proper functioning ofbiological membranes. At physiological temperatures, polar glycerolipidsthat contain only saturated fatty acids cannot form theliquid-crystalline bilayer that is the fundamental structure ofbiological membranes. The introduction of an appropriate number ofdouble bonds (a process referred to as desaturation) into the fattyacids of membrane glycerolipids decreases the temperature of thetransition from the gel to the liquid-crystalline phase and providesmembranes with necessary fluidity. Fluidity of the membrane is importantfor maintaining the barrier properties of the lipid bilayer and for theactivation and function of certain membrane bound enzymes. There is alsoevidence that unsaturation confers some protection to ethanol andoxidative stress, suggesting that the degree of unsaturation of membranefatty acids has importance beyond temperature adaptation. Unsaturatedfatty acids are also precursors of polyunsaturated acids (PUFAs)arachidonic and eicosapentaenoic acids in animals, which are importantsources of prostaglandins. These molecules are local hormones that alterthe activities of the cells in which they are synthesized and inadjoining cells, mediating processes in reproduction, immunity,neurophysiology, thermobiology, and ion and fluid transport.

The ability of cells to modulate the degree of unsaturation in theirmembranes is primarily determined by the action of fatty aciddesaturases. Desaturase enzymes introduce unsaturated bonds at specificpositions in their fatty acyl chain substrates, using molecular oxygenand reducing equivalents from NADH (or NADPH) to catalyze the insertionof double bonds. In many systems, the reaction uses a short electrontransport chain consisting of NAD(P)H, cytochrome b5 reductase, andcytochrome b5, to shuttle electrons from NAD(P)H and the carbon-carbonsingle bond to oxygen, forming water and a double bond (C═C). Manycukaryotic dcsaturases are endoplasmic reticulum (ER) bound non-hemediiron-oxo proteins which contain three conserved histidine-rich motifsand two long stretches of hydrophobic residues. These hydrophobic alphahelical domains are thought to position the protein with its bulkexposed to the cytosolic face of the ER and to organize the active sitehistidines to appropriately coordinate the active diiron-oxo moiety.

While most eukaryotic organisms, including mammals, can introduce adouble bond into an 18-carbon fatty acid at the Δ9 position, mammals areincapable of inserting double bonds at the Δ12 or Δ15 positions. Forthis reason, linoleate (18:2 Δ9,12) and linolenate (18:3 Δ9,12,15) mustbe obtained from the diet and; thus, are termed essential fatty acids.These dietary fatty acids come predominately from plant sources, sinceflowering plants readily desaturate the Δ12 and the Δ15 positions.Certain animals, including some insects and nematodes, can synthesize denovo all their component fatty acids including linolcate and linolenate(Watts and Browse (2002) Proc Natl Acad Sci USA, 99(9):5854-9; Borgesonet al. (1990) Biochim Biophys Acta. 1047(2):135-40; Cripps et al. (1990)Arch Biochem Biophys. 278(1):46-51). The nematode C. elegans, forexample, can synthesize de novo a broad range of polyunsaturated fattyacids including arachidonic acid and eicosapentacnoic acids, a featurenot shared by either mammals or flowering plants (Tanaka et al. (1999)Eur J. Biochem. 263(1):189-95).

The C. elegans desaturase gene fat-2 has been expressed in S. cerevisiaeand shown to be a Δ12 fatty acid desaturase. This enzyme introduces adouble bond between the 12^(th) and the 13^(th) carbons (from thecarboxylate end) and can convert the mono-unsaturated oleate (18:1 Δ9)and palmitoleate (16:1 Δ9) to the di-unsaturated linoleate (18:2 Δ9,12)and 16:2 Δ9,12 fatty acids, respectively.

The nematode Δ12 enzymes are potentially good targets for anti-nematodecompounds for several reasons. Firstly, the enzymes appear to bephylogenetically diverged from their homologs in plants, having lessthan 40% pairwise sequence identity at the amino acid level andphylogenetic analyses demonstrate clustering of nematode Δ12 and ω-3desaturases away from homologs in plants. Experiments with bothtransgenic Arabidopsis and soybeans reveal that plants can toleratesignificant reductions in Δ12 fatty acid desaturase activity, suggestingthat inhibitors of desaturases would likely not be toxic to plants(Singh et al. (2000) Biochem. Society Trans. 28: 940-942; Lee et al.(1998) Science 280:915-918). In addition, as mentioned above, mammalsare thought not to have A 12 fatty acid desaturases. Thus, inhibitors ofthe enzyme are likely to be non-toxic to mammals. Importantly, asdetailed herein, a fatty acid desaturase of nematodes has been shown tobe essential to their viability, both through inhibitor and RNA-mediatedinterference studies. Thus, Δ12 fatty acid desaturases could serve asideal targets for anti-nematode control, as inhibitors of the enzymecould specifically target nematodes while leaving their animal and planthosts unharmed.

Numerous analogs of fatty acids exist and some may act as specificinhibitors of enzymes such as desaturases that act on fatty acids, afact that could be exploited for development of anti-nematode compounds.Sterculic acid, a cyclopropenoid fatty acid analog of oleic acid, is apotent inhibitor of Δ9 fatty acid desaturases (Schmid & Patterson (1998)Lipids 23(3):248-52; Waltermann & Steinbuchel (2000) FEMS MicrobiolLett. 190(1):45-50). It has also been speculated that cyclopropenoidanalogs of linoleic acid may similarly inhibit Δ12 fatty aciddesaturases (Dulayymi et al. (1997) Tetrahedron 53(3): 1099-1110). It isworth noting however that malvalate, a Δ8 cyclopropene fatty acid, seemsto be equally inhibitory to 69 desaturases in some systems, (Schmid &Patterson (1998) Lipids 23(3):248-52), demonstrating how difficult it isto predict inhibitory profiles for some fatty acid analogs. Thia fattyacid analogs (i.e., sulfur containing fatty acids) are also potentialinhibitors of fatty acid desaturases (Skrede et al. (1997) BiochimBiophys Acta 1344(2): 115-131; Hovik et al. (1997) Biochim Biophys Acta1349(3):251-256) as are trans fatty acids (Choi et al. (2001) BiochemBiophys Res Commun 284(3):689-93). However, the specificity andpesticidal activity of these analogs is again difficult to predict(Beach et al. (1989) Mol Biochem Parasitol 35(1):57-66).

Certain fatty acids are also specific receptor antagonists (Yagaloff(1995) Prostaglandins Leukot Essent Fatty Acids 52(5):293-7).

Other analogs of linoleic acid that may also be specific Δ12 inhibitorsinclude the epoxy fatty acid (vernolic acid), the acetylenic fatty acid(crepenynic acid), 12-oxo-9(Z)-octadecenoic acid methyl ester or thehydroxy fatty acids (ricinoleic and ricinelaidic acid). Inhibitors thatinterfere with Δ12 fatty acid desaturase activity are expected to betoxic to nematodes. Importantly, fatty acid analogs such as ricinoleic,ricinelaidic, vernolic and crepenynic acid methyl esters do not appearto be toxic (or are very much less toxic) to at least some plants andare predicted not to be toxic (or are very much less toxic) to at leastsome animals, including mammals. Such fatty acid analogs couldpotentially be used in the development of nematode control agents.

Although previously expressed in plants, fatty acid analogs such ascrepcnynate, ricinoleate and vernolate acids were not thought to bespecific inhibitors of the endogenous plant Δ12 desaturase (Broun &Somerville (1997) Plant. Physiol. 113:933-942; Singh et al. (2000)Biochem. Society Trans. 28(6): 940-942). Changes in the ratio of oleateto linoleate in plants expressing the genes for these analogs wasinstead attributed to a negative interaction between the enzymesinvolved (Singh et al. (2001) Planta 212: 872-879). Addition ofricinoleate exogenously to Nervospora crassa results in a significantdecrease in oleate (C18:1) and an increase in linolenate (C18:3) againproviding no indication that compounds like ricinoleate were in factspecific Δ12 desaturase inhibitors (Goodrich-Tanrikulu et al. (1996)Appl Microbiol Biotechnol. 46(4):382-7).

We made the surprising discovery that methyl esters of certain fattyacid analogs (e.g., ricinoleate, vernolate) are nematicidal and haveactivity consistent with that of specific inhibitors of nematode Δ12desaturases. The fatty acid methyl esters show significantly enhancedactivity over other eighteen carbon fatty acid esters such as oleate,elaidate and linolcate. In contrast to short chain seeminglynon-specific pesticidal fatty acid esters such as laurate andpelargonate, the fatty acid analogs that are predicted Δ12 desaturaseinhibitors show dramatically reduced phytoxicity and can therefore beused effectively while minimizing undesirable damage to non-targetorganisms.

Fatty acid-based analogs or other types of inhibitors may be supplied toplants exogenously, through sprays for example. It is also possible toprovide inhibitors through a host organism or an organism on which thenematode feeds. For example, a host cell that does not naturally producean inhibitor of the M. incognita, H. glycines, D. immitis and S.stercoralis fatty acid desaturase-like polypeptides can be transformedwith enzymes capable of making inhibitory analogs and provided withappropriate precursor chemicals exogenously. Alternatively, the activeinhibitors and precursors can be made endogenously by the expression ofthe appropriate enzymes. In addition, yeast or other organisms can bemodified to produce inhibitors. Nematodes that feed on such organismswould then be exposed to the inhibitors.

In one embodiment, transgenic cells and/or organisms could be generatedthat produce enzymes active on fatty acids (e.g., desaturating,hydroxylating, conjugating, and/or epoxygenating enzymes). Such enzymesmay be expressed, for example, in plants, vertebrates, and/or nematodes.These enzymes may produce fatty acids, analogs, or other inhibitors thatcan then act as specific inhibitors for other enzymes such as a fattyacid desaturase (e.g., a Δ12 epoxygenase from Crepis palaestina producesvernolic acid, a Δ12 desaturase inhibitor, in transgenic Arabidopsis)(Singh et. al. (2000) Biochem. Society Trans. 28:940-942; Lee et al.(1998) Science 280:915-918).

More generally, a recombinant expression vector capable of expressing anenzyme active on fatty acids could be transformed into a host cell of anorganism that is parasitized by a parasitic nematode, (M. incognita, H.glycines. D. immitis or S. stercoralis, for example). Fatty acid analogsthat act as inhibitors of (M. incognita, H. glycines, D. immitis or S.stercoralis Δ12 fatty acid desaturases, for example, can then beproduced in the host cell or organism. In this manner, Δ12 fatty aciddesaturases from feeding parasitic nematodes (e.g., M. incognita, H.glycines, D. immitis or S. stercoralis) could be rendered inactive bythe fatty acid analog.

In another embodiment, a recombinant expression vector harboring a Δ12fatty acid desaturase-like polypeptide from, for example, M. incognita,H. glycines, D. immitis, S. stercoralis or R. axei, can be used toproduce a recombinant fatty acid desaturase polypeptide that isfunctional in a cell, plant or animal, and that can desaturate fattyacids that are normally produced by the cell, plant or animal or thatare provided exogenously to the cell, plant or animal to thecorresponding Δ12 fatty acid. In this way, a cell, plant or animal canbe produced that has a higher proportion of Δ12 unsaturated fatty acidsthan an otherwise similar cell, plant, or animal lacking the recombinantfatty acid desaturase polypeptide. In this way, a cell, plant or animalthat has increased resistance to a Δ12 fatty acid desaturase inhibitorcan be produced.

A recombinant Δ12 fatty acid desaturase, e.g., a M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase,may also be useful for producing lipids having a higher proportion ofΔ12 unsaturated fatty acids, whether by means of recombinant expressionin a cell or in an industrial process using purified nematode Δ12 fattyacid desaturase polypeptide. Such lipids are useful as food oils, asnutritional supplements, and as chemical feedstocks, for example.

The following examples are therefore to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLES

TBLASTN searches of a database of nematode EST sequences (McCarter etal. (1999) Washington University Nematode EST Project) with C. elegansΔ12 fatty acid desaturase (FAT-2) gene sequence (GenBank® Accession No:AAF63745: ID No: 7546993) identified two EST's (AW783527 and AW871151)that are predicted to encode at least a portion of a Δ12 Fatty AcidDesaturase-like enzymes (Δ12 FAT) in one nematode species, M. incognita.

Full Length Δ12 Fatty Acid Desaturase-like cDNA Sequences

The plasmid clone corresponding to the M. incognita EST sequenceAW783527 was obtained from the Genome Sequencing Center (St. Louis,Mo.). This plasmid clone was designated Div249. The cDNA insert in theplasmid was sequenced in its entirety. Unless otherwise indicated, allnucleotide sequences determined herein were sequenced with an automatedDNA sequencer (such as model 373 from Applied Biosystems, Inc.) usingprocesses well known to those skilled in the art. Primers used forsequencing are listed in Table 1 (see below). Partial sequence data forthe M. incognita Δ12 FAT was obtained from Div249, including nucleotidesequence for codons 90-397 and additional 3′ untranslated sequence. Theclone lacked the first 89 codons of the M. incognita Δ12 FAT, as well asthe 5′ untranslated region.

The following methods were used to obtain the full-length nematode Δ12FAT gene and to determine its complete sequence. First, RNA was obtainedfrom plant parasitic nematodes, which are maintained on greenhouse potcultures depending on nematode preference. Root Knot Nematodes(Meloidogyne sp) were propagated on Rutgers tomato (Burpee). Total RNAwas isolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml ofpacked worms were combined with 8 ml TRIZOL reagent and solubilized byvortexing. Following 5 minutes of incubation at room temperature, thesamples are divided into smaller volumes and spun at 14,000×g for 10minutes at 4° C. to remove insoluble material. The liquid phase wasextracted with 200 μl of chloroform, and the upper aqueous phase wasremoved to a fresh tube. The RNA was precipitated by the addition of 500μl of isopropanol and centrifuged to pellet. The aqueous phase wascarefully removed, and the pellet was washed in 75% ethanol and spun tore-collect the RNA pellet. The supernatant was carefully removed, andthe pellet was air dried for 10 minutes. The RNA pellet was resuspendedin 50 μl of DEPC-H₂0 and analyzed by spectrophotometry at 260 and 280 nmto determine yield and purity. Yields could be 1-4 mg of total RNA from2 ml of packed worms.

To obtain the missing 5′ sequence of the M. incognita Δ12 FAT gene, the5′ RACE technique was applied, and SL1 PCR was performed using firststrand cDNA from M. incognita as a template. Briefly, SL1 PCR utilizesthe observation, that unlike most eukaryotic mRNAs, many nematode mRNAmolecules contain a common leader sequence (5′ ggg ttt aat tac cca agtttg a 3′; SEQ ID NO: 17) transpliced to their 5′ ends. If this sequenceis present on the 5′ end of a cDNA, that cDNA can be amplified using PCRwith a primer that binds to the SL1 transpliced leader and agene-specific primer near the 3′ end of the cDNA.

Briefly, following the instructions provided by Life Technologies cDNAsynthesis kit, first strand cDNA synthesis was performed on totalnematode RNA using SuperScript™ II Reverse Transcriptase and an oligo-dTprimer (which anneals to the natural poly A tail found on the 3′ end ofall eukaryotic mRNA). RNase H was then used to degrade the original mRNAtemplate. Following degradation of the original mRNA template, the firststrand cDNA was directly PCR amplified without further purificationusing Taq DNA polymerase, a gene specific primer (FAT10, SEQ ID NO: 19)designed from known sequence that anneals to a site located within thefirst strand cDNA molecule, and the SL1 primer, which is homologous the5′ end of the cDNA of interest. Amplified PCR products were then clonedinto a suitable vector for DNA sequence analysis. This procedure wasperformed to obtain clone Div864. This clone contained codons 1-120 inaddition to 5′ untranslated sequences. Taken together, clones Div249 andDiv864 contain sequences comprising the complete open reading frame ofthe Δ12 FAT gene from M. incognita.

To obtain the complete Δ12 fatty acid desaturase gene from M. incognitaon one clone, primers FAT30 and FAT31 were designed to amplify thecomplete open reading frame. Following PCR amplification, severalindependent clones were obtained. DNA sequence analysis revealed thattwo very similar but distinct fatty acid desaturase genes had beenidentified. Along with the gene sequence reported for SEQ ID NO: 1 (1191nucleotide ORF, 397 amino acid polypeptide, FIG. 1), we also identifiedSEQ ID NO: 2 (1239 nucleotide ORF, 413 amino acid polypeptide, FIG. 2).The two genes are very similar and are identical in the regionshomologous to primers FAT30 and FAT31. Clones containing sequencesidentical to SEQ ID NO: 1 included Div1456 and Div1459. Clonescontaining sequences identical to SEQ ID NO: 2 included Div1458 andDiv1463. While cloning the first two M. incognita Δ12 fatty aciddesaturase genes, a third Δ12 fatty acid desaturase gene fragment fromM. incognita was obtained by PCR amplification using the FAT10/SL1primer combination, resulting in clone Div866. This clone containscodons 1-108 of the third M. incognita Δ12 fatty acid desaturase gene.In order to obtain the complete gene sequence, a gene-specific primer(FAT23, SEQ ID NO: 23) designed to a known sequence that anneals to asite located within the first strand cDNA molecule, and an oligo dTprimer, which is homologous to the 3′ end of the cDNA of interest wereused. Amplified PCR products were then cloned into a suitable vector forDNA sequence analysis. This procedure was performed to obtain cloneDiv2727. This clone contains codons 96-387 in addition to 3′untranslated sequences. Taken together, clones Div866 and Div2727contain sequences comprising the complete open reading of the third Δ12fatty acid desaturase gene from M. incognita. The gene sequence reportedfor SEQ ID NO: 3 (1161 nucleotide ORF, 387 amino acid polypeptide, FIG.3) is very similar to the first two M. incognita Δ12 fatty aciddesaturase genes. The first two predicted Δ12 fatty acid desaturasepolypeptides (SEQ ID NO: 8 and 9) are approximately 92% identical toeach other and approximately 57% and 56% identical to the C. elegansfatty acid desaturase (SEQ ID NO: 32), respectively, and areapproximately 69% and 70% identical to the third M. incognita predictedΔ12 fatty acid desaturase polypeptide (SEQ ID NO: 10), respectively. Thethird predicted Δ12 FAT polypeptide (SEQ ID NO: 10) is approximately 51%identical to the C. elegans Δ12 fatty acid desaturase (SEQ ID NO: 32)and 69% similar.

In order to obtain the H. glycines Δ12 fatty acid desaturase gene, the5′ RACE technique was applied, and SL1 PCR was performed using firststrand cDNA from H. glycines as a template (cDNA synthesis explainedabove). The first strand cDNA was directly PCR amplified using the SL1primer and a gene specific degenerate primer (FAT33, SEQ ID NO: 24)designed to anneal to region of strong homology shared across manynematode desaturase genes. Amplified PCR products were then cloned intoa suitable vector for DNA sequence analysis. This procedure wasperformed to obtain clone Div1870. This clone contained codons 1-193 inaddition to 5′ untranslated sequences. To obtain the 3′ sequence of thegene, the 3′ RACE technique was applied . The first strand cDNA wasdirectly PCR amplified using a gene specific primer (FAT44, SEQ ID NO:25) designed from known sequence that anneals within the first strandcDNA molecule of interest, and an oligo dT primer, which is homologousto the 3′ end of the cDNA of interest. This procedure was performed togenerate clone Div2724, which contains codons 144-389 in addition to 3′untranslated sequences. Taken together, clones Div1870 and Div2724contain sequences comprising the complete open reading frame of the Δ12fatty acid desaturase gene of H. glycines. The predicted Δ12 fatty aciddesaturase polypeptide reported for SEQ ID NO: 11, (1167 nucleotide ORF,389 amino acid polypeptide, FIG. 4) is approximately 56% identical and73% similar to the C. elegans fatty acid desaturase (SEQ ID NO: 32).

In an attempt to obtain the D. immitis Δ12 fatty acid desaturase gene,first strand cDNA, using D. immitis cDNA as a template (cDNA synthesisdescribed above), was directly PCR amplified, using a gene-specificdegenerate primer (FAT35, SEQ ID NO: 26) designed to anneal to a regionof strong homology shared across many nematode desaturase genes, andanother gene-specific degenerate primer (FT05, SEQ ID NO: 27), which waspredicted to be homologous to a region near the 5′ end of the cDNA ofinterest. Amplified PCR products were then cloned into a suitable vectorfor DNA sequence analysis. This procedure was performed to obtain cloneDiv3228, which contains codons 1-224, which correspond to codons 78-301of the C. elegans fatty acid desaturase (SEQ ID NO: 32). To acquire themissing 3′ sequence of D. immitis Δ12 fatty acid desaturase, the 3′ RACEtechnique was applied using a gene-specific primer (FAT 12, SEQ ID NO:28) designed to a known sequence that anneals to a site located withinthe first strand cDNA molecule, and an oligo dT primer, which ishomologous to the 3′ end of the cDNA of interest. Amplified PCR productswere then cloned into a suitable DNA vector for sequence analysis. Thisprocedure was performed to obtain the clone Div3230, which contained thecodons 196-289, which correspond to codons 273-376 of the C. elegansfatty acid desaturase (SEQ ID NO: 32), in addition to 3′ untranslatedsequences. Taken together, clones Div3228 and Div3230 compriseapproximately 80% of the complete D. immitis Δ12 FAT open reading frame.The 5′ end sequence of this gene has yet to be completed. The partialΔ12 fatty acid desaturase polypeptide sequence reported for SEQ ID NO:12 (867 nucleotide ORF, 289 amino acid polypeptide, FIG. 5) is 62%identical and 75% similar to the C. elegans fatty acid desaturase (SEQID NO: 32).

Plasmid clone, Div3013, corresponding to the S. stercoralis EST sequence(GenBank® Identification No: 9830288) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Full sequence data for the S. stercoralis Δ12fatty acid desaturase was obtained from Div3013, including nucleotidesequence for codons 1-368, (the full open reading frame) and additional5′ and 3′ untranslated sequences. The predicted gene sequence reportedfor SEQ ID NO: 6 (1104 nucleotide ORF, 368 amino acid polypeptide, FIG.6) is approximately 61% identical and 76% similar to the C. elegansfatty acid desaturase (SEQ ID NO: 32).

In order to obtain the sequence of the R. axei Δ12 fatty acid desaturasegene, first strand cDNA from R. axei was directly PCR amplified, using agene-specific degenerate primer (FAT35, SEQ ID NO: 26) designed toanneal to region of strong homology shared across many nematodedesaturase genes, and another gene-specific degenerate primer (FAT34,SEQ ID NO: 29) designed to anneal to region of strong homology sharedacross many nematode desaturase genes near the 3′ end of the cDNA ofinterest. Amplified PCR products were then cloned into a suitable DNAvector for sequence analysis. This procedure was performed to obtain theclone Div1843, which contained the codons 185-301 (an internal fragmentmissing both the 5′ and 3′ ends of the gene). In order to obtain themissing 5′ sequence of the R. axei Δ12 fatty acid desaturase gene, the5′ RACE technique was applied and SL1 PCR was performed using firststrand cDNA from R. axei as a template (cDNA synthesis described above).The first strand cDNA was directly PCR amplified using a gene specificprimer (FAT40, SEQ. ID. NO. 30) designed from the known sequence ofclone Div 1843, that anneals to a site located within the cDNA ofinterest, and the SL primer, which is homologous to the 5′ end of manynematode cDNAs. Amplified PCR products were then cloned into a suitablevector for DNA sequence analysis. This procedure was performed to obtainthe clone Div2026, which contained the codons 1-199, in addition to 5′untranslated sequences. To obtain the missing 3′ sequence of the gene,the 3′ RACE technique was applied using a gene specific primer (FAT42,SEQ ID NO: 31) designed from the known sequence of clone Div1843, thatanneals to a site located within the cDNA of interest, and an oligo dTprimer, which is homologous to the 3′ end of the cDNA of interest.Amplified PCR products were then cloned into a suitable vector for DNAsequence analysis. This procedure was performed to obtain clone Div2149.This clone contained codons 246-374 in addition to 3′ untranslatedsequences. Taken together, clones Div1843, Div2149, and Div2026 containsequences comprising the complete open reading frame of the Δ12 FAT genefrom R. axei. The predicted Δ12 fatty acid desaturase polypeptide genesequence reported for SEQ ID NO: 7, (1122 nucleotide ORF, 374 amino acidpolypeptide, FIG. 7) is approximately 71% identical and 82% similar toC. elegans fatty acid desaturase (SEQ ID NO: 32).

TABLE 2 Primers Employed in Cloning SEQ ID Name Sequence NO: Homology toT7 Gtaatacgactcactatagggc 15 vector polylinker primer T3Aattaaccctcactaaaggg 16 vector polylinker primer SL1Gggtttaattacccaagtttga 17 nematode transpliced leader Oligo dTgagagagagagagagagagaactagtctcgagtttttttttttttttttt 18 universal primerto poly A tail FAT10 5′ aag ttc cgt gcc cac aat c 3′ 19 Mi Δ12 FAT(codons 115-120)* FAT11 5′ gcc aaa aat gag aac cat cg 3′ 20 Mi Δ12 FAT(codons 206-211)* FAT30 5′ atg tct tat ctt gac aca ac 3′ 21 Mi Δ12 FAT(codons 1-6)* FAT31 5′ cta ttt atc ctt ttt att at 3′ 22 Mi Δ12 FAT(codons 392-397)* FAT23 tctatattcgctgttggacacg 23 Mi Δ12 FAT (codons96-102) FAT33 gtrtadatnggrttcca 24 Ce Δ12 FAT (codons 174-179) FAT44ctggtactgcttgctcggca 25 Hg Δ12 FAT (codons 92-97) FAT35aaraaraartgrtgngcnacrtg 26 Ce Δ12 FAT (codons 295-30l)^(#) FT05atgggnatgttyggntc 27 Ce Δ12 FAT (codons 81-85)^(#) FT12gtacaaaccattgatcgag 28 Di Δ12 FAT (codons 196-201) FAT34gayggntctcayttytggccntgg 29 Ce Δ12 FAT (codons 185-192 )^(#) FAT40ctctatcttcagtcgttgtg 30 Ra Δ12 FAT (codons 179-202) FAT42ggttatcatcacctatctgc 31 Ra Δ12 FAT (codons 246-251) *codon numbering isbased on SEQ ID NO:1. ^(#)codon numbering is based on SEQ ID NO:32Characterization of M. incognita, H. glycines, D. immitis. S.stercoralis and R. axei Δ12 Fatty Acid Desaturase Genes.

The similarity between the Δ12 fatty acid desaturase proteins from M.incognita, H. glycines, D. immitis, S. stercoralis and R. axei from C.elegans is presented as a multiple alignment generated by the ClustalXmultiple alignment program as described below (FIG. 8).

The similarity between M. incognita, H. glycines, D. immitis, S.stercoralis and R. axei Δ12 fatty acid desaturase-like sequences andother sequences was also investigated by comparison to sequencedatabases using BLASTP analysis against nr (a non-redundant proteinsequence database available on the Internet at www.ncbi.nlm.nih.gov) andTBLASTN analysis against dbest (an EST sequence database available onthe Internet at www.ncbi.nlm.nih.gov; top 500 hits; E=1e-4). The “Expect(E) value” is the number of sequences that are predicted to align bychance to the query sequence with a score S or greater given the size ofthe database queried. This analysis was used to determine the potentialnumber of plant and vertebrate homologs for each of the nematode fattyacid desaturase-like polypeptides described above. While M. incognita(SEQ ID NO: 1, 2 and SEQ ID NO:3), H. glycines (SEQ ID NO:4), D. immitis(SEQ ID NO:5), S. stercoralis (SEQ ID NO:6) and R. axei (SEQ ID NO:7)fatty acid desaturase-like cDNA sequences had numerous plant hits, theyhad no vertebrate hits in nr or dbest having sufficient sequencesimilarity to meet the threshold E value of 1e-4 (this E valueapproximately corresponds to a threshold for removing sequences having asequence identity of less than about 25% over approximately 100 aminoacids). Accordingly, the M. incognita, H. glycines, D. immitis, S.stercoralis and R. axei fatty acid desaturase-like enzymes of thisinvention do not appear to share significant sequence similarity withthe more common vertebrate fatty acid desaturase enzymes such as the Δ6fatty acid desaturase of Homo sapiens (a member of the FAD family ofdesaturases, GenBank Accession No. NP_(—)068373), the Δ5 fatty aciddesaturase of Homo sapiens (also a member of the FAD family, GenBankAccession No. NP_(—)037534) or other mammalian fatty acid dcsaturases.

On the basis of the lack of similarity to vertebrates, the D. immitisand S. stercoralis fatty acid desaturase-like enzymes (e.g., Δ12 fattyacid desaturase) are useful targets of inhibitory compounds selectivefor some nematodes over their hosts (e.g., humans, animals). While atleast some plants have fatty acid desaturases that are somewhathomologous to those in parasitic nematodes, including M. incognita andH. glycines, other criteria make them promising targets for the controlof plant parasitic nematodes, including the fact that at least in somecases, plants can tolerate significant reductions in fatty aciddesaturase activity while, as demonstrated below, nematodes can not.

Functional predictions were made using four iterations of PSI-BLAST withthe default parameters on the nr database. PSI-BLAST searches andmultiple alignment construction with CLUSTALX demonstrated that the C.elegans gene (GenBank® Accession No: AAF63745) was a member of the fattyacid desaturase family. Reciprocal blast searches and phylogenetic treesconfirm that the nucleotide sequences in M. incognita, H. glycines, D.immitis, S. stercoralis and R. axei do encode orthologs of the C.elegans gene and therefore are also likely fatty acid desaturaseproteins with the same activity. Protein localizations were predictedusing the TargetP server available on the Internet at:www.cbs.dtu.dk/services/TargetP. The M. incognita fatty acid desaturase(SEQ ID NO: 8, 9 and SEQ ID NO:10), H. glycines (SEQ ID NO:11), D.immitis (SEQ ID NO:12), S. stercoralis (SEQ ID NO:13) and R. axei (SEQID NO: 14) polypeptides are likely to be cytoplasmic. In addition, theyare predicted to have four transmembrane regions, consistent with themodel that they are membrane bound.

RNA Mediated Interference (RNAi)

A double stranded RNA (dsRNA) molecule can be used to inactivate a Δ12fatty acid desaturase (Δ12 FAT) gene in a cell by a process known as RNAmediated-interference (Fire ct al. (1998) Nature 391:806-811, and Gönezyet al. (2000) Nature 408:331-336). The dsRNA molecule can have thenucleotide sequence of a Δ12 FAT nucleic acid described herein or afragment thereof. The dsRNA molecule can be delivered to nematodes viadirect injection, or by soaking nematodes in aqueous solution containingconcentrated dsRNA, or by raising bacteriovorous nematodes on E. coligenetically engineered to produce the dsRNA molecule.

RNAi by injection: To examine the effect of inhibiting Δ12 FAT activity,a Δ12 dsRNA was injected into the nematode, basically as described inMello et al. (1991) EMBO J. 10:3959-3970. Briefly, a plasmid wasconstructed that contains a portion of the C. elegans Δ12 FAT genesequence, specifically a fragment 651 nucleotides long, containing theentire first exon and terminating just before the conserved intronsplice junction between the first exon and first intron. This constructencodes approximately the first 217 amino acids of the C. elegans Δ12FAT gene. Primers were used to specifically amplify this sequence as alinear dsDNA. Single-stranded RNAs were transcribed from these fragmentsusing T7 RNA polymerase and SP6 RNA polymerase (the RNAs correspond tothe sense and antisense RNA strands). RNA was precipitated andresuspended in RNAse-free water. For annealing of ssRNAs to form dsRNAs,ssRNAs were combined, heated to 95° for two minutes then allowed to coolfrom 700 to room temperature over 1.5-2.5 hours.

DsRNA was injected into the body cavity of 15-20 young adult C. eleganshermaphrodites. Worms were typically immobilized on an agarose pad andinjected with 2-5 μl of dsRNA at a concentration of 1 mg/ml. Injectionswere performed with visual observation using a Zeiss Axiovert compoundmicroscope equipped with 10× and 40×DIC objectives, for example. Needlesfor microinjection were prepared using a Narishige needle puller, stagemicromanipulator (Leitz) and a N₂-powered injector (Narishige) set at10-20 p.s.i. After injection, 200 μl of recovery buffer (0.1% salmonsperm DNA, 4% glucose, 2.4 mM KCl, 66 mM NaCl, 3 mM CaCl₂, 3 mM HEPES,pH 7.2) were added to the agarose pad and the worms were allowed torecover on the agarose pad for 0.5-4 hours. After recovery, the wormswere transferred to NGM agar plates seeded with a lawn of E. coli strainOP50 as a food source. The following day and for 3 successive daysthereafter, 7 individual healthy injected worms were transferred to newNGM plates seeded with OP50. The number of eggs laid per worm per dayand the number of those eggs that hatch and reach fertile adulthood weredetermined. As a control, Green Fluorescent Protein (GFP) dsRNA wasproduced and injected using similar methods. GFP is a commonly usedreporter gene originally isolated from jellyfish and is widely used inboth prokaryotic and eukaryotic systems. The GFP gene is not present inthe wild-type C. elegans genome and, therefore, GFP dsRNA does nottrigger an RNAi phenotype in wild-type C. elegans. The C. clegans Δ12FAT RNAi injection phenotype presented as a strongly reduced F1hatch-rate, with the few surviving individuals arrested in an earlylarval stage.

RNAi by feeding: C. elegans can be grown on lawns of E. coli geneticallyengineered to produce double stranded RNA (dsRNA) designed to inhibitΔ12 FAT expression. Briefly, E. coli were transformed with a genomicfragment of a portion of the C. elegans Δ12 FAT gene sequence,specifically a fragment 651 nucleotides long, containing the entirefirst exon and terminating just before the conserved intron splicejunction between the first exon and first intron. This construct encodesapproximately the first 217 amino acids of the C. clegans Δ12 FAT gene.The 651 nucleotide genomic fragment was cloned into an E. coliexpression vector between opposing T7 polymerase promoters. The clonewas then transformed into a strain of E. coli that carries anIPTG-inducible T7 polymerase. As a control, E. coli was transformed witha gene encoding the Green Fluorescent Protein (GFP).

Feeding RNAi was initiated from C. elegans eggs or from C. elegans L4s.When feeding RNAi was started from C. elegans eggs at 23° C. on NGMplates containing IPTG and E. coli expressing the C. elegans Δ12 FAT orGFP dsRNA, the C. elegans Δ12 FAT RNAi feeding phenotype presented aspartially sterile F1 individuals and dead F2 embryos. When feeding RNAiwas started from C. elegans L4 larvae at 23° C. on NGM plates containingIPTG and E. coli expressing the C. elegans Δ12 FAT or GFP dsRNA, the C.elegans RNAi feeding phenotype presented as partially sterile P₀individuals with developmentally arrested, sterile F1 nematodes.

C. elegans cultures grown in the presence of E. coli expressing dsRNAand those injected with dsRNA from the Δ12 FAT gene were stronglyimpaired indicating that the fatty acid desaturase-like gene provides anessential function in nematodes and that dsRNA from the fatty aciddesaturase-like gene is lethal when ingested by or injected into C.elegans.

Rescue of C. elegans Δ12 FAT RNAi Feeding Phenotype by Linoleic AcidMethyl Ester

The C. elegans Δ12 fatty acid desaturase (Fat2 protein) converts themono-unsaturated oleic acid to the di-unsaturated fatty acid linoleicacid. The 612 FAT RNAi prevents expression of the Δ12 fatty aciddesaturase, which is predicted to cause a decrease in levels of linoleicacid in the nematode, leading to arrested development and death.Addition of 3 mM linoleic acid methyl ester to the NGM media used forthe RNAi experiment brings about a partial rescue of the Δ12 FAT RNAifeeding phenotype. Addition of 3 mM oleic acid methyl ester does notrescue the Δ12 FAT RNAi feeding phenotype (see Table 3 below).

TABLE 3 C. elegans Δ12 FAT RNAi feeding phenotypes (starting with C.elegans L4 larvae as the P₀ animal) Fatty Acid Added P₀ phenotype F1phenotype F2 phenotype None Severely reduced Developmentally NA egglaying arrested and sterile (almost sterile) Oleic Acid Severely reducedDevelopmentally NA Methyl Ester egg laying arrested and sterile (almoststerile) Linoleic Acid Reduced egg Moderately delayed Slightly MethylEster laying development and delayed moderately reduced development egglayingInhibitor Studies

Vernolic acid and ricinoleic acid are naturally occurring plant-producedfatty acid homologs that we predict to be specific inhibitors of Δ12 FATenzymes. The addition of these compounds to living cultures of C.elegans is expected to mimic the effects of the Δ12 FAT RNAi experimentssince, in each case, the phenotype observed should derive from theinhibition of the nematode Δ12 FAT. To explore this possibility, C.elegans cultures were started from eggs on NGM plates containing theirE. coli food source and one of either 3 mM ricinolcic acid methyl esteror 3 mM vernolic acid methyl ester. Total eggs layed and hatch-rates ofF1 and F2 individuals were followed and compared to nematode culturesgrown in the presence of control fatty acids oleic acid methyl ester andlinoleic acid methyl ester. C. elegans L4 larvae were added to NGMplates containing OP50 E. coli and one of the following methyl esters:oleic acid, linolcic acid, ricinoleic acid, vernolic acid or none. C.elegans L4 larvae growing on plates containing ricinoleic acid (FIG. 11)or vernolic acid (FIG. 12) methyl esters developed to mature adults moreslowly than those on control plates containing oleic acid (FIG. 9) orlinoleic acid (FIG. 10) and produced very few embryos (eggs). Of theembryos that hatched, the young larvae displayed severe arrestedphenotypes and did not develop to adults (FIGS. 11 and 12). C. eleganscultures growing on plates containing no fatty acid methyl esters oroleic acid or linoleic acid methyl esters exhibited no dramaticlifecycle impairments (FIGS. 9 and 10).

Identification of Additional Fatty Acid Desaturase-Like Sequences

A skilled artisan can utilize the methods provided in the example aboveto identify additional nematode fatty acid desaturase-like sequences,e.g., fatty acid desaturase-like sequences (including Δ12 fatty aciddesaturase sequences) from nematodes other than M. incognita, H.glycines, D. immitis, S. stercoralis, R. axei and/or C. elegans. Inaddition, nematode fatty acid desaturase-like sequences can beidentified by a variety of methods including computer-based databasesearches, hybridization-based methods, and functional complementation.

Database Identification A nematode fatty acid desaturase-like sequencecan be identified from a sequence database, e.g., a protein or nucleicacid database using a sequence disclosed herein as a query. Sequencecomparison programs can be used to compare and analyze the nucleotide oramino acid sequences. One such software package is the BLAST suite ofprograms from the National Center for Biotechnology Institute (NCBI;Altschul et al. (1997) Nuc. Acids Research 25:3389-3402). A fatty aciddesaturase-like sequence of the invention can be used to query asequence database, such as nr, dbest (expressed sequence tag (EST)sequences), and htgs (high-throughput genome sequences), using acomputer-based search, e.g., FASTA, BLAST, or PSI-BLAST search.Homologous sequences in other species (e.g., humans and animals) can bedetected in a PSI-BLAST search of a database such as nr (E value=10, Hvalue=1e-2, using, for example, four iterations;http://www.ncbi.nlm.nih.gov/). Sequences so obtained can be used toconstruct a multiple alignment, e.g., a ClustalX alignment, and/or tobuild a phylogenetic tree, e.g., in ClustalX using the Neighbor-Joiningmethod (Saitou et al. (1987) Mol. Biol. Evol. 4:406-425) andbootstrapping (1000 replicates; Felsenstein (1985) Evolution39:783-791). Distances may be corrected for the occurrence of multiplesubstitutions [D_(corr)=−ln(1-D-D²/5) where D is the fraction of aminoacid differences between two sequences](Kimura (1983) The Neutral Theoryof Molecular Evolution, Cambridge University Press).

The aforementioned search strategy can be used to identify fatty aciddesaturase-like sequences in nematodes of the fobllowing non-limiting,exemplary genera:

Plant Parasitic Nematode Genera Include:

Afrina, Anguina, Aphelenchoides, Belonolaimus, Bursaph elenchus,Cacopaurus, Cactodera, Criconema, Criconemoides, Cryphodera,Ditylenchus, Dolichodorus, Dorylaimus, Globodera, Helicotylenchus,Hemicriconemoides, Hemicycliophora, Heerodera, Hirschmanniella,Hoplolaimus, Hypsoperine, Longidorus, Meloidogvne, Mesoanguina,Nacobbus, Nacobbodera, Panagrellus, Paratrichodorus, Paratylenchus,rarylenchus, Pterorylenchus, Punctodera, Radopholus, Rhadinaphelenchus,Rotylecnchulus, Rotylenchus, Scutellonemna, Subanguina,Thecavermiculatus, Trichodorus, Turbatrix, Tylenchorhynchus,Tylenchulus, Xiphinema.

Animal and Human Parasitic Nematode Genera Include:

Acanthocheilonema, Aelurostrongylus, Ancylostoma, Angiostrongylus,Anisakis, Ascaris, Ascarops, Bunostomunm, Brugia, Capillaria, Chabertia,Cooperia, Crenosoma, Cyalthostonme species (Small Strongyles),Dictyocaulus, Dioctophyma, Dipetalonema, Dirofiliaria, Dracunculus,Draschia, Elaneophora, Enterobius, Filaroides, Gnathostoma, Gonylonema,Habronema, Haemonchus, Hyostrongylus, Lagochilascaris, Litomosoides,Loa, Mammomonogamus, Mansonella, Muellerius, Metastrongylid, Necator,Nemiatodirus, Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca,Ostertagia, Oxyspirura, Oxyuris, Parafilaria, Parascaris,Parastrongyloides, Parelaphostrongylus, Physaloptera, Physocephalus,Protostrongyhlus, Pseudoterranova, Setaria, Spirocerca, Stephanurus,Stephanofilaria, Strongyloides, Strongylus, Spirocerca, Syngamus,Teladorsagia, Thelazia, Toxascaris, Toxocara, Trichinella,Trichostrongylus, Trichuris, Uncinaria, and Wuchereria.

Particularly Preferred Nematode Genera Include:

Plant parasitic: Anguina, Aphelenchoides, Belonolaimus, Bursaphelenchus.Ditylenchus, Dolichodorus, Globodera, Heterodera, Hoplolaimus,Longidorus, Meloidogyne, Nacobbus, Pratylenchus, Radopholus,Rotylenchus, Tylenchulus, Xiphinema.

Animal & Human parasitic: Ancylostoma, Ascaris, Brugia, Capillaria,Cooperia, Cyathostome species, Diclyocaulus, Dirofiliaria, Dracunculus,Enterobius, Haenonchus, Necator, Nematodirus, Oesophagostomumn,Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides,Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella,Trichostrongylus, Trickhuris, and Wuchereria.

Particularly Preferred Nematode Species include:

Plant parasitic: Anguina tritici, Aphelenchoides fragariae, Belonolaimuslongicaudattus, Bursaphelenchus xylophilus, Ditylenchus destructor,Ditylenchus dipsaci, Dolichodorus heterocephalous, Globodera pallida,Globodera rostochiensis, Globodera tabacum, Heterodera avenae,Heterodera cardiolata, Heterodera carotae, Heterodera cruciferae,Heterodera glycines, Heterodera major, Heterodera schachtii, Heteroderazeae, Hoplolaimus tylenchiformis, Longidorus sylphus, Meloidogyneacronea, Meloidogyne arenaria, Meloidogyne chitwoodi, Meloidogyneexigua, Meloidogyne graminicola, Meloidogyne hapla, Meloidogyneincognita, Meloidogyne javanica, Meloidogyne nassi, Nacobbusbatatiformis, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae,Radopholus similis, Rotylenchus reniformis, Tvlenchulus semipenetrans,Xiphinema americanum.

Animal & Human parasitic: Ancylostoma braziliense, Ancylostoma caninutm,Ancylostoma cevlanicunm, Ancylostoma duodenale, Ancylostomna tubaefbrme,Ascaris suum, Ascaris lumbrichoides, Brugia malayi, Capillaria bovis,Capillaria plica, Capillaria feliscati, Cooperia oncophora, Cooperiapunctata, Cyathostome species, Dictyocaulus filaria, Dictyocaulusviviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis, Dracunculusinsignis, Enterobius vermicularis, Halemonchus contortus, Haemonchusplacei, Necator amnericanus, Nematodirus helvetianus, Oesophagostomumradiatum, Onchocerca volvulus, Onchocerca cervicalis, Ostertagiaostertagi, Ostertagia circurmcincta, Oxvuris equi, Parascaris equortum,Strongyloides stercoralis, Strongylus vulgaris, Strongylus edentatlus,Syngamus trachea, Teladorsagia circumcincta, Toxocara cati, Trichinellaspiralis, Trichostrongylus axei, Trichostrongylus colubriformis,Trichuris vulpis, Trichuris suis, Trichurs trichiura, and Wuchereriabancrofti.

Further, a fatty acid desaturase-like sequence can be used to identifyadditional fatty acid desaturase-like sequence homologs within a genome.Multiple homologous copies of a fatty acid desaturase-like sequence canbe present. For example, a nematode fatty acid desaturase-like sequencecan be used as a seed sequence in an iterative PSI-BLAST search (defaultparameters, substitution matrix=Blosum62, gap open=11, gap extend=1) ofa nonredundant database such as wormpep (E value=1e-2, H value=1e-4,using, for example 4 iterations) to determine the number of homologs ina database, e.g., in a database containing the complete genome of anorganism. A nematode fatty acid desaturase-like sequence can be presentin a genome along with 1, 2, 3, 4, 5, 6, 8, 10, or more homologs.

Hybridization Methods A nematode fatty acid desaturase-like sequence canbe identified by a hybridization-based method using a sequence providedherein as a probe. For example, a library of nematode genomic or cDNAclones can be hybridized under low stringency conditions with the probenucleic acid. Stringency conditions can be modulated to reducebackground signal and increase signal from potential positives. Clonesso identified can be sequenced to verify that they encode fatty aciddesaturase-like sequences.

Another hybridization-based method utilizes an amplification reaction(e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g.,degenerate oligonucleotides, are designed to hybridize to a conservedregion of a fatty acid desaturase-like sequence. The oligonucleotidesare used as primers to amplify a fatty acid desaturase-like sequencefrom template nucleic acid from a nematode, e.g., a nematode other thanM. incognita, and/or C. elegans. The amplified fragment can be clonedand/or sequenced.

Complementation Methods A nematode fatty acid desaturase-like sequencecan be identified from a complementation screen for a nucleic acidmolecule that restores fatty acid desaturase-like activity to a celllacking a fatty acid desaturase-like activity. Routine methods can beused to construct strains (i.e., nematode, yeast, bacterial strains)that lack fatty acid desaturase activity. For example, a nematode strainmutated at the fatty acid desaturase gene locus can be grown (i.e.,rescued) on supplements such as Δ12 unsaturated fatty acids. Such astrain can be transformed with nematode cDNAs predicted to encode fattyacid desaturases. Strains can be identified in which fatty aciddesaturase activity is restored by selecting for those transgenic linesthat exhibit growth in the absensce of supplemental Δ12 unsaturatedfatty acids. The plasmid harbored by the rescued strain can be recoveredto identify and/or characterize the inserted nematode cDNA that providesfatty acid desaturase-like activity when expressed. Similarly, abacterial and/or yeast strain can be used as a selection system, wherebythe Δ12 fatty acid desaturase gene(s) can be mutated using, for example,phage transduction (Clark et al. (1983) Biochem. 22:5897-5902; Simon etal. (1980) J. Bacteriology 142:621-632). The mutant cell line can besustained on exongenous unsaturated fatty acids. A strain lacking Δ12fatty acid desaturase gene(s) can be transformed with a plasmid libraryexpressing nematode cDNAs. Strains can be identified in which fatty aciddesaturase activity is restored, i.e., that can grow in the absence ofexogenous fatty acids. In still another embodiment, a microorganism(i.e., a yeast strain) that naturally does not contain a Δ12 fatty aciddesaturase can be transformed with plasmids expressing nematode genes.Transformed strains that exhibit Δ12 fatty acid desaturase activity canbe identified using, GC analysis to measure fatty acid composition.Those clones that convert oleic acid to linoleic acid can be selected,for example (Sakurdani et al. (1999) Eur. J. Biochem. 261:812-820.

Full-length cDNA and Sequencing Methods The following methods can beused, e.g., alone or in combination with another method describedherein, to obtain full-length nematode fatty acid desaturase-like genesand determine their sequences.

Plant parasitic nematodes are maintained on greenhouse pot culturesdepending on nematode preference. Root Knot Nematodes (Meloidogyne sp)are propagated on Rutgers tomato (Burpee), while Soybean Cyst Nematodes(Heterodera sp) are propagated on soybean. Total nematode RNA isisolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packedworms are combined with 8 ml TRIZOL reagent and solubilized byvortexing. Following 5 minutes of incubation at room temperature, thesamples are divided into smaller volumes and spun at 14,000×g for 10minutes at 4° C. to remove insoluble material. The liquid phase isextracted with 200 μl of chloroform, and the upper aqueous phase isremoved to a fresh tube. The RNA is precipitated by the addition of 500μl of isopropanol and centrifuged to pellet. The aqueous phase iscarefully removed, and the pellet is washed in 75% ethanol and spun tore-collect the RNA pellet. The supernatant is carefully removed, and thepellet is air dried for 10 minutes. The RNA pellet is resuspended in 50μl of DEPC-H₂0 and analyzed by spectrophotometry at λ260 and 280 nm todetermine yield and purity. Yields can be 1-4 mg of total RNA from 2 mlof packed worms.

Full-length cDNAs can be generated using 5′ and 3′ RACE techniques incombination with EST sequence information. The molecular technique 5′RACE (Life Technologies, Inc., Rockville, Md.) can be employed to obtaincomplete or near-complete 5′ ends of cDNA sequences for nematode fattyacid desaturase-like cDNA sequences. Briefly, following the instructionsprovided by Life Technologies, first strand cDNA is synthesized fromtotal nematode RNA using Murine Leukemia Virus Reverse Transcriptase(M-MLV RT) and a gene specific “antisensc” primer, e.g., designed fromavailable EST sequence. RNase H is used to degrade the original mRNAtemplate. The first strand cDNA is separated from unincorporated dNTPs,primers, and proteins using a GlassMAX Spin Cartridge. Terminaldeoxynucleotidyl transferase (TdT) is used to generate a homopolymericdC tailed extension by the sequential addition of dCTP nucleotides tothe 3′ end of the first strand cDNA. Following addition of the dChomopolymeric extension, the first strand cDNA is directly amplifiedwithout further purification using Taq DNA polymerase, a gene specific“antisense” primer designed from available EST sequences to anneal to asite located within the first strand cDNA molecule, and adeoxyinosine-containing primer that anneals to the homopolymeric dCtailed region of the cDNA in a polymerase chain reaction (PCR). 5′ RACEPCR amplification products are cloned into a suitable vector for furtheranalysis and sequencing.

The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville,Md.), can be employed to obtain complete or near-complete 3′ ends ofcDNA sequences for nematode fatty acid desaturase-like cDNA sequences.Briefly, following the instructions provided by Life Technologies(Rockville, Md.), first strand cDNA synthesis is performed on totalnematode RNA using SuperScript™ Reverse Transcriptase and an oligo-dTprimer that anneals to the polyA tail. Following degradation of theoriginal mRNA template with RNase H, the first strand cDNA is directlyPCR amplified without further purification using Taq DNA polymerase, agene specific primer designed from available EST sequences to anneal toa site located within the first strand cDNA molecule, and a “universal”primer which contains sequence identity to 5′ end of the oligo-dTprimer. 3′ RACE PCR amplification products are cloned into a suitablevector for further analysis and sequencing.

Nucleic Acid Variants

Isolated nucleic acid molecules of the present invention include nucleicacid molecules that have an open reading frame encoding a fatty aciddesaturase-like polypeptide. Such nucleic acid molecules includemolecules having: the sequences recited in SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7; and sequences coding for the fatty acid desaturase-likeproteins recited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. Thesenucleic acid molecules can be used, for example, in a hybridizationassay to detect the presence of a M. incognita, H. glycines, D. immitis,S. stercoralis or R. axei nucleic acid in a sample.

The present invention includes nucleic acid molecules such as thoseshown in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 that may be subjected tomutagenesis to produce single or multiple nucleotide substitutions,deletions, or insertions. Nucleotide insertional derivatives of thenematode gene of the present invention include 5′ and 3′ terminalfusions as well as intra-sequence insertions of single or multiplenucleotides. Insertional nucleotide sequence variants are those in whichone or more nucleotides are introduced into a predetermined site in thenucleotide sequence, although random insertion is also possible withsuitable screening of the resulting product. Deletion variants arecharacterized by the removal of one or more nucleotides from thesequence. Nucleotide substitution variants are those in which at leastone nucleotide in the sequence has been removed and a differentnucleotide inserted in its place. Such a substitution may be silent(e.g., synonymous), meaning that the substitution does not alter theamino acid defined by the codon. Alternatively, substitutions aredesigned to alter one amino acid for another amino acid (e.g.,non-synonymous). A non-synonymous substitution can be conservative ornon-conservative. A substitution can be such that activity, e.g., fattyacid desaturase-like activity, is not impaired. A conservative aminoacid substitution results in the alteration of an amino acid for asimilar acting amino acid, or amino acid of like charge, polarity, orhydrophobicity, e.g., an amino acid substitution listed in Table 3below. At some positions, even conservative amino acid substitutions candisrupt the activity of the polypeptide.

TABLE 4 Conservative Amino Acid Replacements For Amino Acid Code Replacewith any of . . . Alanine Ala Gly, Cys, Ser Arginine Arg Lys, HisAsparagine Asn Asp, Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln CysteineCys Met, Thr, Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp,Asn, Gln Glycine Gly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu,Met Leucine Leu Val, Ile, Met Lysine Lys Arg, His Methionine Met Ile,Leu, Val Phenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser Thr,Cys, Ala Threonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr TyrosineTyr Phe, His Valine Val Leu, Ile, Met

The current invention also embodies splice variants of nematode fattyacid desaturase-like sequences.

Another aspect of the present invention embodies a polypeptide-encodingnucleic acid molecule that is capable of hybridizing under conditions oflow stringency (or high stringency) to the nucleic acid molecule putforth in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 or their complements.

The nucleic acid molecules that encode for fatty acid desaturase-likepolypeptides may correspond to the naturally occurring nucleic acidmolecules or may differ by one or more nucleotide substitutions,deletions, insertions, and/or additions. Thus, the present inventionextends to genes and any functional mutants, derivatives, parts,fragments, naturally occurring polymorphisms, homologs or analogsthereof or non-functional molecules. Such nucleic acid molecules can beused to detect polymorphisms of fatty acid desaturase genes or fattyacid desaturase-like genes, e.g., in other nematodes. As mentionedbelow, such molecules are useful as genetic probes; primer sequences inthe enzymatic or chemical synthesis of the gene; or in the generation ofimmunologically interactive recombinant molecules. Using the informationprovided herein, such as the nucleotide sequence SEQ ID NO: 1, 2, 3, 4,5, 6 and/or 7, a nucleic acid molecule encoding a fatty aciddesaturase-like molecule may be obtained using standard cloning and ascreening techniques, such as a method described herein.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, or in the form of DNA, including, for example, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. The nucleic acids may be inthe form of RNA/DNA hybrids. Single-stranded DNA or RNA can be thecoding strand, also referred to as the sense strand, or the non-codingstrand, also known as the anti-sense strand.

Expression of Fatty Acid Desaturase-Like Polypeptides

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes at least one isolated nucleic acidmolecule depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7, inserted in avector capable of delivering and maintaining the nucleic acid moleculeinto a cell. The DNA molecule may be inserted into an autonomouslyreplicating factor (suitable vectors include, for example, pGEM3Z andpcDNA3, and derivatives thereof). The vector nucleic acid may be abacteriophage DNA such as bacteriophage lambda or M13 and derivativesthereof. The vector may be either RNA or DNA, single- ordouble-stranded, prokaryotic, eukaryotic, or viral. Vectors can includetransposons, viral vectors, episomes, (e.g., plasmids), chromosomesinserts, and artificial chromosomes (e.g. BACs or YACs). Construction ofa vector containing a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include, but are not limited to, F. coli. Suitable eukaryotichosts include yeast such as S. cerevisiae, other fungi, vertebratecells, invertebrate cells (e.g., insect cells), plant cells, humancells, human tissue cells, and whole eukaryotic organisms. (e.g., atransgenic plant or a transgenic animal). Further, the vector nucleicacid can be used to generate a virus such as vaccinia or baculovirus.

The present invention also extends to genetic constructs designed forpolypeptide expression. Generally, the genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences. A variety of transcriptional control sequences arewell known to those in the art and may be functional in, but are notlimited to, a bacterium, yeast, plant, or animal cell. The expressionvector can also include a translation regulatory sequence (e.g., anuntranslated 5′ sequence, an untranslated 3′ sequence, a poly A additionsite, or an internal ribosome entry site), a splicing sequence orsplicing regulatory sequence, and a transcription termination sequence.The vector can be capable of autonomous replication or it can integrateinto host DNA.

In an alternative embodiment, the DNA molecule is fused to a reportergene such as β-glucuronidase gene, β-galactosidase (lacZ),chloramphenicol-acetyltransferase gene, a gene encoding greenfluorescent protein (and variants thereof), or red fluorescent proteinfirefly lucifcrase gene, among others. The DNA molecule can also befused to a nucleic acid encoding a polypeptide affinity tag, e.g.glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag orreporter fusion joins the reading frames of SEQ ID NO: 1, 2, 3, 4, 5, 6and/or 7 to the reading frame of the reporter gene encoding the affinitytag such that a translational fusion is generated. Expression of thefusion gene results in translation of a single polypeptide that includesboth a nematode fatty acid desaturase-like region and a reporter proteinor affinity tag. The fusion can also join a fragment of the readingframe of SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7. The fragment can encode afunctional region of the fatty acid desaturase-like polypeptides, astructurally intact domain, or an epitope (e.g., a peptide of about 8,10, 20, or or more amino acids). A nematode fatty acid desaturase-likenucleic acid that includes at least one of a regulatory region (e.g., a5′ regulatory region, a promoter, an enhancer, a 5′ untranslated region,a translational start site, a 3′ untranslated region, a polyadenylationsite, or a 3′ regulatory region) can also be fused to a heterologousnucleic acid. For example, the promoter of a fatty acid desaturase-likenucleic acid can be fused to a heterologous nucleic acid, e.g., anucleic acid encoding a reporter protein.

Suitable cells to transform include any cell that can be transformedwith a nucleic acid molecule of the present invention. A transformedcell of the present invention is also herein referred to as arecombinant or transgenic cell. Suitable cells can either beuntransformed cells or cells that have already been transformed with atleast one nucleic acid molecule. Suitable cells for transformationaccording to the present invention can either be: (i) endogenouslycapable of expressing the fatty acid desaturase-like protein or; (ii)capable of producing such protein after transformation with at least onenucleic acid molecule of the present invention.

In an exemplary embodiment, a nucleic acid of the invention is used togenerate a transgenic nematode strain, e.g., a transgenic C. elegansstrain. To generate such a strain, nucleic acid linked to a C. eleganspromoter is injected into the gonad of a nematode, thus generating aheritable extrachromosomal array containing the nucleic acid (see, e.g.,Mello et al. (1991) EMBO J. 10:3959-3970). The transgenic nematode canbe propagated to generate a strain harboring the transgene. To identifyspecific inhibitors of the M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei A12 FAT2, the C. elegans Δ12 FAT gene can be“knocked out” by continuous growth on E. coli engineered to producedsRNA homologous to the C. elegans Δ12 FAT gene (described earlier).Nematodes of the strain can be used in screens to identify inhibitorsspecific for a M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like gene.

In another embodiment, a nucleic acid of the invention can be clonedbehind a yeast-specific transcription promoter can be used to generate atransgenic yeast strain, such as Saccharomyces cerevisiae. The S.cerevisiae strain can be transformed using the lithium acetate procedure(Ito et al. (1983) J. Bacteriology 153:163-168; Sakuradani (1999) Eur J.Biochem. 261:812-820). Such a strain can be used to identify inhibitorsspecific for a M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like polypeptide.

Production of Fatty Acid Desaturase-Like Polypeptide Substrates andInhibitors

In still another embodiment, a nucleic acid of the invention can be usedto generate a transgenic plant such as Arrabidopis thaliana, a modellegume Medicago truncatula, or any plant of interest, e.g., a nematodehost. For example, the fatty acid desaturase like-gene can be clonedinto a vector under the control of the cauliflower mosaic virus (CaMV)35S promoter/nopaline synthase terminator cassette (Baulcombe et al.(1986) Nature 321:446-449) which can then be introduced into anAgrobacterium strain by the freeze thaw method. Agrobacterium-mediatedtransformation can be accomplished by the planta-vacuum-infiltrationmethod (Bouchez et al. (1993) C. R. Acad. Sci. Paris 316:1188-1193) andtransformed transgeneic plant lines can be selected (Spychalla et al.(1997) Proc. Natl. Acad. Sci. 94:1142-1147). Such a plant line can beused to identify inhibitors specific for M. incognita, H. glycines, D.immitis, S. stercoralis or R. axei fatty acid desaturase-likepolypeptides or other plant fatty acid desaturase-like polypeptides.Fatty acid desaturase can be expressed in soybean and/or soybean somaticembryos using, for example, particle bombardment method oftransformation (Finer et al. (1991) In Vitro Cell. Dev. Biol.27:175-182; Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96:12935-12940). It is also desirable to generate plants with increasedresistance to inhibitors of fatty acid desaturase-like polypeptides byproviding the plant with a transgene expressing a fatty acid desaturase.A transformed cell that harbors a M. incognita fatty acid desaturasepolypeptide may naturally produce products of Δ12 fatty acid desaturases(e.g. linoleic acid). In this circumstance, the cell may produce ahigher proportion of Δ12-desaturated fatty acids than an otherwisesimilar cell lacking the M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase polypeptide.

If the host cell does not naturally produce a substrate for fatty aciddesaturase, one or more substrates can be provided exogenously to cellstransformed with an expressible fatty acid desaturase polynucleotide(e.g., by topical application (Spychalla et al. (1997) Proc. Natl. Acad.Sci. USA 94:1142-1147)), or fatty acid desaturase can be co-expressed incells together with one or more cloned genes that encode polypeptidesthat can produce substrate compounds from precursor compounds in suchcells.

A transformed cell may also be engineered that harbors a polypeptidethat produces inhibitors of the Δ12 fatty acid desaturase-like gene ofnematodes. For example, a cell may be engineered to produce a Δ12hydroxylase, a Δ12 acetylenase, and/or a Δ12 epoxygenase.

Such polypeptides may produce fatty acid analogs that inhibit thenematode Δ12 fatty acid desaturase-like polypeptide (i.e., ricinolcicacid, crepenynic acid, and vernolic acid). Such genes may be linked to aroot-specific promoter and transformed into a plant, for example.Examples of suitable genes include: Crepsis palacstina Δ12 fatty acidepoxygenase (GenBank® Accession No. CAA76156; produces vernolic acid);Crepis alpina Δ12 fatty acid acetylenase (GenBank® Accession No.CAA76158; produces crepenynic acid); Ricinus Communis oleate12-hydroxylase (GenBank® Accession No. AAC49010; produces ricinoleicacid); Momordica charantia Δ12 oleic desaturase-like protein (GenBank®Accession No. AAF05916; produces alpha-eleostearic acid); and Impatiensbalsamina Δ12 oleic acid desaturase-like protein (GenBank® Accession No.AAF05915; produces alpha-eleostearic acid).

Oligonucleotides

Also provided are oligonucleotides that can form stable hybrids with anucleic acid molecule of the present invention. The oligonucleotides canbe about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80,100, 120 nucleotides in length. The oligonucleotides can be used asprobes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit nematode fatty aciddesaturase-like protein activity or production (e.g., antisense, triplexformation, ribozyme, and/or RNA drug-based reagents). The presentinvention includes oligonucleotides of RNA (ssRNA and dsRNA), DNA, orderivatives of either. The invention extends to the use of sucholigonucleotides to protect non-nematode organisms (for example e.g.,plants and animals) from disease by reducing the viability of infectingnamatodes, e.g., using a technology described herein. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto a non-nematode organism using techniques known to those skilled inthe art, including, but not limited to, transgenic expression in plantsor animals.

Primer sequences can be used to amplify a fatty acid desaturase-likenucleic acid or fragment thereof. For example, at least 10 cycles of PCRamplification can be used to obtain such an amplified nucleic acid.Primers can be at least about 8-40, 10-30 or 14-25 nucleotides inlength, and can anneal to a nucleic acid “template molecule”, e.g., atemplate molecule encoding a fatty acid desaturase-like geneticsequence, or a functional part thereof, or its complementary sequence.The nucleic acid primer molecule can be any nucleotide sequence of atleast 10 nucleotides in length derived from, or contained withinsequences depicted in SEQ ID NO: 1, 2, 3, 4, 5, 6 and/or 7 and theircomplements. The nucleic acid template molecule may be in a recombinantform, in a virus particle, bacteriophage particle, yeast cell, animalcell, plant cell, fungal cell, or bacterial cell. A primer can bechemically synthesized by routine methods.

This invention embodies any fatty acid desaturase-like sequences thatare used to identify and isolate similar genes from other organisms,including nematodes, prokaryotic organisms, and other eukaryoticorganisms, such as other animals and/or plants.

In another embodiment, the invention provides oligonucleotides that arespecific for a M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like nucleic acid molecule. Sucholigonucleotides can be used in a PCR test to determine if a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei nucleicacid is present in a sample, e.g., to monitor a disease caused M.incognita, H. glycines, D. immitis or S. stercoralis.

Protein Production

Isolated fatty acid desaturase-like proteins from nematodes can beproduced in a number of ways, including production and recovery of therecombinant proteins and/or chemical synthesis of the protein. In oneembodiment, an isolated nematode fatty acid desaturase-like protein isproduced by culturing a cell, e.g., a bacterial, fungal, plant, oranimal cell, capable of expressing the protein, under conditions foreffective production and recovery of the protein. The nucleic acid canbe operably linked to a heterologous promoter, e.g., an induciblepromoter or a constitutive promoter. Effective growth conditions aretypically, but not necessarily, in liquid media comprising salts, water,carbon, nitrogen, phosphate sources, minerals, and other nutrients, butmay be any solution in which fatty acid desaturase-like proteins may beproduced.

In one embodiment, recovery of the protein may refer to collecting thegrowth solution and need not involve additional steps of purification.Proteins of the present invention, however, can be purified usingstandard purification techniques, such as, but not limited to, affinitychromatography, thermaprecipitation, immunoaffinity chromatography,ammonium sulfate precipitation, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, and others.

The fatty acid desaturase-like polypeptide can be fused to an affinitytag, e.g., a purification handle (e.g., glutathione-S-reductase,hexa-histidine, maltose binding protein, dihydrofolate reductases, orchitin binding protein) or an epitope tag (e.g., c-myc epitope tag,FLAG™ tag, or influenza HA tag). Affinity tagged and epitope taggedproteins can be purified using routine art-known methods.

Antibodies Against Fatty Acid Desaturase-like Polypeptides

Recombinant fatty acid desaturase-like gene products or derivativesthereof can be used to produce immunologically interactive molecules,such as antibodies, or functional derivatives thereof. Useful antibodiesinclude those that bind to a polypeptide that has substantially the samesequence as the amino acid sequences recited in SEQ ID NO: 8, 9, 10, 11,12, 13 and/or 14, or that has at least 80% similarity over 50 or moreamino acids to these sequences. In a preferred embodiment, the antibodyspecifically binds to a polypeptide having the amino acid sequencerecited in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14. The antibodies canbe antibody fragments and genetically engineered antibodies, includingsingle chain antibodies or chimeric antibodies that can bind to morethan one epitope. Such antibodies may be polyclonal or monoclonal andmay be selected from naturally occurring antibodies or may bespecifically raised to a recombinant fatty acid desaturase-like protein.

Antibodies can be derived by immunization with a recombinant or purifiedfatty acid desaturase-like gene or gene product. As used herein, theterm “antibody” refers to an immunoglobulin, or fragment thereof.Examples of antibody fragments include F(ab) and F(ab′)₂ fragments,particularly functional ones able to bind epitopes. Such fragments canbe generated by proteolytic cleavage, e.g., with pepsin, or by geneticengineering. Antibodies can be polyclonal, monoclonal, or recombinant.In addition, antibodies can be modified to be chimeric, or humanized.Further, an antibody can be coupled to a label or a toxin.

Antibodies can be generated against a full-length fatty aciddesaturase-like protein, or a fragment thereof, e.g., an antigenicpeptide. Such polypeptides can be coupled to an adjuvant to improveimmunogenicity. Polyclonal serum is produced by injection of the antigeninto a laboratory animal such as a rabbit and subsequent collection ofsera. Alternatively, the antigen is used to immunize mice. Lymphocyticcells are obtained from the mice and fused with myelomas to formhybridomas producing antibodies.

Peptides for generating fatty acid desaturase-like antibodies can beabout 8, 10, 15, 20, 30 or more amino acid residues in length, e.g., apeptide of such length obtained from SEQ ID NO: 8, 9, 10, 11, 12, 13and/or 14. Peptides or epitopes can also be selected from regionsexposed on the surface of the protein, e.g., hydrophilic or amphipathicregions. An epitope in the vicinity of the active or binding site can beselected such that an antibody binding such an epitope would blockaccess to the active site or prevent binding. Antibodies reactive with,or specific for, any of these regions, or other regions or domainsdescribed herein are provided. An antibody to a fatty aciddesaturase-like protein can modulate a fatty acid desaturase-likeactivity.

Monoclonal antibodies, which can be produced by routine methods, areobtained in abundance and in homogenous form from hybridomas formed fromthe fusion of immortal cell lines (e.g., myclomas) with lymphocytesimmunized with fatty acid desaturase-like polypeptides such as those setforth in SEQ ID NO: 8, 9, 10, 11, 12, 13 and/or 14.

In addition, antibodies can be engineered, e.g., to produce a singlechain antibody (see, for example, Colcher et al. (1999) Ann N Y Acad Sci880: 263-80; and Reiter (1996) Clin Cancer Res 2: 245-52). In stillanother implementation, antibodies are selected or modified based onscreening procedures, e.g., by screening antibodies or fragments thereoffrom a phage display library.

Antibodies of the present invention have a variety of important useswithin the scope of this invention. For example, such antibodies can beused: (i) as therapeutic compounds to passively immunize an animal inorder to protect the animal from nematodes susceptible to antibodytreatment; (ii) as reagents in experimental assays to detect presence ofnematodes; (iii) as tools to screen for expression of the gene productin nematodes, animals, fungi, bacteria, and plants; and/or (iv) as apurification tool of fatty acid desaturase-like protein; (v) as fattyacid desaturase inhibitors/activators that can be expressed orintroduced into plants or animals for therapeutic purposes.

An antibody against a fatty acid desaturase-like protein can be producedin a plant cell, e.g., in a transgenic plant or in culture (see, e.g.,U.S. Pat. No. 6,080,560).

Antibodies that specifically recognize a M. incognita. H. glycines, D.immitis, S. stercoralis or R. axei fatty acid desaturase-like proteinscan be used to identify a M. incognita. H. glycines, D. immitis, S.stercoralis or R. axei nematodes, and, thus, can be used to monitor adisease caused by M. incognita, H. glycines, D. immitis or S.stercoralis.

Nucleic Acids Agents

Also featured are isolated nucleic acids that are antisense to nucleicacids encoding nematode fatty acid desaturase-like proteins. An“antisense” nucleic acid includes a sequence that is complementary tothe coding strand of a nucleic acid encoding a fatty aciddesaturase-like protein. The complementarity can be in a coding regionof the coding strand or in a noncoding region, e.g., a 5′ or 3′untranslated region, e.g., the translation start site. The antisensenucleic acid can be produced from a cellular promoter (e.g., a RNApolymerase II or III promoter), or can be introduced into a cell, e.g.,using a liposome. For example, the antisense nucleic acid can be asynthetic oligonucleotide having a length of about 10, 15, 20, 30, 40,50, 75, 90, 120 or more nucleotides in length.

An antisense nucleic acid can be synthesized chemically or producedusing enzymatic reagents, e.g., a ligase. An antisense nucleic acid canalso incorporate modified nucleotides, and artificial backbonestructures, e.g., phosphorothioate derivative, and acridine substitutednucleotides.

Ribozymes: The antisense nucleic acid can be a ribozyme. The ribozymecan be designed to specifically cleave RNA, e.g., a fatty aciddesaturase-like mRNA. Methods for designing such ribozymes are describedin U.S. Pat. No. 5,093,246 or Haselhoffand Gerlach (1988) Nature334:585-591. For example, the ribozyme can be a derivative ofTetrahymena L-19 IVS RNA in which the nucleotide sequence of the activesite is modified to be complementary to a fatty acid desaturase-likenucleic acid (see, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cechet al. U.S. Pat. No. 5,116,742).

Peptide Nucleic acid (PNA): An antisense agent directed against a fattyacid desaturase-like nucleic acid can be a peptide nucleic acid (PNA).See Hyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23) formethods and a description of the replacement of the deoxyribosephosphate backbone for a pseudopeptide backbone. A PNA can specificallyhybridize to DNA and RNA under conditions of low ionic strength as aresult of its electrostatic properties. The synthesis of PNA oligomerscan be performed using standard solid phase peptide synthesis protocolsas described in Hyrup et al. (1996) supra and Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-675.

RNA Mediated Interference (RNAi): A double stranded RNA (dsRNA) moleculecan be used to inactivate expression of a fatty acid desaturase-likegene in a cell by a process known as RNA mediated-interference (RNAi;e.g., Fire et al. (1998) Nature 391:806-811, and Gönezy et al. (2000)Nature 408:331-336). The dsRNA molecule can have the nucleotide sequenceof a fatty acid desaturase-like nucleic acid described herein or afragment thereof. The molecule can be injected into a cell, or asyncitia, e.g., a nematode gonad as described in Fire et al., supra.

In one embodiment, dsRNA can be introduced by soaking of nematodes.Double-stranded RNA may be introduced to nematodes by directly soakingindividual worms in a solution of dsRNA. Soaking of C. elegans in asolution of dsRNA can be accomplished essentially as described in Tabaret al. ((1998) Science 282:430-1)). Briefly, Hermaphrodite L4-stage C.elegans are washed twice in siliconized tubes with approximately 1 ml M9buffer (5 g/L NaCl, 11.32 g/L Na₂ HPO₄.7H₂O, 3 g/L KH₂ PO₄, 1 mM MgSO₄).In each wash, the worms are allowed to settle for 10 minutes and most ofthe supernatant removed. Between five and twenty worms in minimal volume(5-10 ul) are transferred to a fresh siliconized tube and an equalvolume of specific dsRNA (resuspended in sterile, RNase-free water) isadded. The final concentration of dsRNA is generally between 0.1 and 3.0mg/ml. Up to 10% (v/v) lipofectin (Gibco-BRL) may be added to the mix.The mixture is incubated for 10 to 30 hours at a constant temperaturebetween 15° and 23° C. The worms are then transferred individually toNGM-agar plates containing a lawn of E. coli (such as strain OP50),incubated at constant temperature between 15° and 23° C. and scoreddaily for phenotypes of the worms and their progeny for at least fourconsecutive days.

Screening Assays

Another embodiment of the present invention is a method of identifying acompound capable of altering (e.g., inhibiting or enhancing) theactivity of fatty acid desaturase-like molecules. This method, alsoreferred to as a “screening assay,” herein, includes, but is not limitedto, the following procedure: (i) contacting an isolated fatty aciddesaturase-like protein with a test inhibitory compound under conditionsin which, in the absence of the test compound, the protein has fattyacid desaturase-like activity; and (ii) determining if the test compoundalters the fatty acid desaturase-like activity or alters the ability ofthe fatty acid desaturase to regulate other polypeptides or moleculese.g., the ability of the enzyme to desaturate fatty acids. Suitableinhibitors or activators that alter a nematode fatty aciddesaturase-like activity include compounds that interact directly with anematode fatty acid desaturase-like protein, perhaps but notnecessarily, in the active or binding site. They can also interact withother regions of the nematode fatty acid desaturase protein by bindingto regions outside of the active site or site responsible forregulation, for example, by allosteric interaction.

In one embodiment, an M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like polypeptide isexpressed in a yeast cell, for example in S. cerevisiae, as has beendescribed for a C. elegans FAT-2-like polypeptide (Peyo-Ndi (2000)Archives of Biochemistry and Biophysics 376:399-408). Overall fatty acidcomposition from wild-type and fatty acid desaturase-harboring yeast canthan be assessed using, for example, gas-chromotography-massspectrometry techniques (GC-MS). Optimally, an increase in Δ12unsaturated fatty acids would be concomitant with introduction of an M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide into the yeast strain. Test compounds canthen be added to the yeast strain and fatty acid composition can bemeasured. A test compound that alters fatty acid composition,particularly decreases Δ12 unsaturated fatty acids in the yeast strainharboring the M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like polypeptides would be consideredcandidate compounds.

In one embodiment, an M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase-like polypeptide isexpressed in a eukaryotic or plant cell, for example in Chinese hamsterovary cells or rabbit skin cells. Overall fatty acid composition fromwild-type and fatty acid desaturase-harboring eukaryotic cells can thanbe assessed using, for example, gas chromatography-mass spectrometrytechniques (GC-MS). Optimally, an increase in Δ12 unsaturated fattyacids would be concomitant with introduction of an M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like polypeptide into the cells. Test compounds can then beadded to the eukaryotic cells and fatty acid composition can bemeasured. A test compound that alters fatty acid composition,particularly decreases Δ12 unsaturated fatty acids in the cellsharboring the M. incognita, H. glycines, D. immitis, S. stercoralis orR. axei fatty acid desaturase-like polypeptides would be consideredcandidate compounds.

Similarly, a M. incognita, H. glycines, D. immitis, S. stercoralis or R.axei fatty acid desaturase-like polypeptides can be expressed in plants,for example in soybean, cotton, tobacco, potato, M. truncatula, and/orArabidopsis. Plants used for transformation may have a functional Δ12fatty acid desaturase gene or may be deficient in Δ12 fatty aciddesaturase activity. Relative amounts of fatty acids can be measured andcompared. Compounds that alter fatty acid compositions, particularlythose that decrease Δ12 unsaturated fatty acids, would be candidatecompounds.

Compounds: A test compound can be a large or small molecule, forexample, an organic compound with a molecular weight of about 100 to10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A testcompound can be any chemical compound, for example, a small organicmolecule, a carbohydrate, a lipid, an amino acid, a polypeptide, anucleoside, a nucleic acid, or a peptide nucleic acid. Small moleculesinclude, but are not limited to, metabolites, metabolic analogues,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (i.e., includingheteroorganic and organometallic compounds). Compounds and componentsfor synthesis of compounds can be obtained from a commercial chemicalsupplier, e.g., Sigma-Aldrich Corp. (St. Louis, Mo.). The test compoundor compounds can be naturally occurring, synthetic, or both. A testcompound can be the only substance assayed by the method describedherein. Alternatively, a collection of test compounds can be assayedeither consecutively or concurrently by the methods described herein. Acompound may be an analog. Specifically, inhibitors may be analogs offatty acids, for example, cyclypropenoid analogs of linoleic acid. Epoxyfatty acids (vernolic acid), acetylenic fatty acids (crepenynic acid)and hydroxy fatty acids (ricinoleic acid) may be used as inhibitors.Analogs such as α-elostearic acid with conjugated double bonds may beacceptable inhibitors. These may be expressed in plants (e.g. a Δ12epoxygenase from C. palaestina produces vernolic acid in transgenicArabidopsis (Singh et. al., supra).

Compounds can also act by allosteric inhibition or by preventing thefatty acid desaturase from binding to, and thus, acting on its target,i.e., a fatty acid.

A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. It may becyclypropenoid analogs of linoleic acid, epoxy fatty acids (vernolicacid), acetylenic fatty acids (crepenynic acid), and/or hydroxy fattyacids (ricinoleic acid). Analogs such as α-elostcaric acid withconjugated double bonds may be acceptable inhibitors. A library can bedesigned and synthesized to cover such classes of chemicals, e.g., asdescribed in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909;Erb et al. (1994) Proc. Natl. Acad. Sci. USA. 91:11422; Zuckennann etal. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303;Carrell et al. (1994) Angew. Chem. Int. Ed. Eng. 33:2059; Carell et al.(1994) Angew Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J.Med. Chem. 37:1233.

Organism-based Assays: Organisms can be grown in microtiter plates,e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates. In oneembodiment, the organism is a nematode. The nematodes can be geneticallymodified. Non-limiting examples of such modified nematodes include: 1)nematodes or nematode cells (M. incognita, H. glycines, D. immitis, S.stercoralis, R. axei, and/or C. elegans) having one or more fatty aciddesaturase-like genes inactivated (e.g., using RNA mediatedinterference); 2) nematodes or nematode cells expressing a heterologousfatty acid desaturase-like gene, e.g., a fatty acid desaturase-like genefrom another species; and 3) nematodes or nematode cells having one ormore endogenous fatty acid desaturase-like genes inactivated andexpressing a heterologous fatty acid desaturase-like gene, e.g., a M.incognita, H. glycines, D. immitis, S. stercoralis or R. axei fatty aciddesaturase-like gene as described herein.

A plurality of candidate compounds, e.g., a combinatorial library, canbe screened. The library can be provided in a format that is amenablefor robotic manipulation, e.g., in microtitre plates. Compounds can beadded to the wells of the microtiter plates. Following compound additionand incubation, viability and/or reproductive properties of thenematodes or nematode cells are monitored.

The compounds can also be pooled, and the pools tested. Positive poolsare split for subsequent analysis. Regardless of the method, compoundsthat decrease the viability or reproductive ability of nematodes,nematode cells, or progeny of the nematodes are considered leadcompounds.

In another embodiment, the compounds can be tested on a microorganism,(e.g., a yeast and/or bacterium). For example, a M. incognita, H.glycines, D. innmmitis, S. stercoralis or R. axei fatty acid desaturasegene can be expressed in S. cerevisiae, as has been described for a C.elegans FAT-2 like polypeptide (Peyo-Ndi (2000) Archives of Biochem andBiophysics 376: 399-408). The generation of such strains is routine inthe art. As described above for nematodes and nematode cells, the celllines can be grown in microtitre plates, each well having a differentcandidate compound or pool of candidate compounds. Growth is monitoredduring or after the assay to determine if the compound or pool ofcompounds is a modulator of a nematode fatty acid desaturase-likepolypeptide.

In another embodiment fatty acid composition of the organisms can bedetermined, using, for example, GC-MS. Fatty acid methyl esters can beformed by a transesterification reaction and extracted. GC-MS can beperformed using a Hewlet Packard 6890 gas chromatograph interfaced witha Hewlet Packard 5973 mass selective detector, for example. Retentiontimes of resulting methyl esters can be compared with those of knownsamples (Cahoon, supra). In another embodiment, ethyl esters can beextracted with hexane and analyzed by GLC through a 50 m by 0.32 mmCP-Wax58-CB fused silica column (Chrompack) (Singh, supra; Lee, supra).In another embodiment, if a M. incognita, H. glycines, D. immitis, S.stercoralis or R. axei fatty acid desaturase gene is expressed in amicroorganism (i.e., yeast), fatty acid methyl esters can be preparedusing methanolic HCl as described (Reed et al. (2000) Plant Physiol.122:715-720; Meesapyodsuk, (2000) Biochemistry 39:11948-11954).

In Vitro Activity Assays: The screening assay can be an in vitroactivity assay. For example, a nematode fatty acid desaturase-likepolypeptide can be purified as described above. The polypeptide can bedisposed in an assay container, e.g., a well of a microtitre plate alongwith an appropriate substrate. A candidate compound can be added to theassay container, and the fatty acid desaturase-like activity ismeasured. Optionally, the activity is compared to the activity measuredin a control container in which no candidate compound is disposed or inwhich an inert or non-functional compound is disposed. Fatty acidcomposition can be determined, using, for example, GC-MS. Fatty acidmethyl esters can be formed by a transesterification reaction andextracted. GC-MS can be performed using a Hewlet Packard 6890 gaschromatograph interfaced with a Hewlet Packard 5973 mass selectivedetector, for example. Retention times of resulting methyl esters can becompared with those of known samples (Cahoon, supra). In anotherembodiment, ethyl esters can be extracted with hexane and analyzed byGLC through a 50 m by 0.32 mm CP-Wax58-CB fused silica column(Chrompack) (Singh, supra; Lee, supra).

In Vitro Binding Assays: The screening assay can also be a cell-freebinding assay, e.g., an assay to identify compounds that bind a nematodefatty acid desaturase-like polypeptide. For example, a nematode fattyacid desaturase-like polypeptide can be purified and labeled. Thelabeled polypeptide is contacted to beads; each bead has a tagdetectable by mass spectroscopy, and test compound, e.g., a compoundsynthesized by combinatorial chemical methods. Beads to which thelabeled polypeptide is bound are identified and analyzed by massspectroscopy. The beads can be generated using “split-and-pool”synthesis. The method can further include a second assay to determine ifthe compound alters the activity of the fatty acid desaturase-likepolypeptide.

Optimization of a Compound: Once a lead compound has been identified,standard principles of medicinal chemistry can be used to producederivatives of the compound. Derivatives can be screened for improvedpharmacological properties, for example, efficacy, pharmacokinetics,stability, solubility, and clearance. The moieties responsible for acompound's activity in the above-described assays can be delineated byexamination of structure-activity relationships (SAR) as is commonlypracticed in the art. One can modify moieties on a lead compound andmeasure the effects of the modification on the efficacy of the compoundto thereby produce derivatives with increased potency. For an example,see Nagarajan et al. (1988) J. Antibiot. 41:1430-8. A modification caninclude N-acylation, amination, amidation, oxidation, reduction,alkylation. esterification, and hydroxylation. Furthermore, if thebiochemical target of the lead compound is known or determined, thestructure of the target and the lead compound can inform the design andoptimization of derivatives. Molecular modeling software is commerciallyavailable (e.g., Molecular Simulations, Inc.). “SAR by NMR,” asdescribed in Shuker et al. (1996) Science 274:1531-4, can be used todesign ligands with increased affinity, by joining lower-affinityligands.

A preferred compound is one that interferes with the function of a fattyacid desaturase-like polypeptide and that is not substantially toxic toplants, animals, or humans. By “not substantially toxic” it is meantthat the compound does not substantially affect the respective animal,or human fatty acid desaturase proteins or fatty acid desaturaseactivity. Thus, particularly desirable inhibitors of M. incognita, H.glycines, D. immitis, S. stercoralis or R. axei fatty acid desaturase donot substantially inhibit fatty acid desaturase-like polypeptides orfatty acid desaturase activity of vertebrates, e.g., humans for example.Other desirable compounds do not substantially inhibit to fatty aciddesaturase activity of plants.

Standard pharmaceutical procedures can be used to assess the toxicityand therapeutic efficacy of a modulator of a fatty acid desaturase-likeactivity. The LD50 (the dose lethal to 50% of the population) and theED50 (the dose therapeutically effective in 50% of the population can bemeasured in cell cultures, experimental plants (e.g., in laboratory orfield studies), or experimental animals. Optionally, a therapeutic indexcan be determined which is expressed as the ratio: LD50/ED50. Hightherapeutic indices are indicative of a compound being an effectivefatty acid desaturase-like inhibitor, while not causing undue toxicityor side-effects to a subject (e.g., a host plant or host animal).

Alternatively, the ability of a candidate compound to modulate anon-nematode fatty acid desaturase-like polypeptide is assayed, e.g., bya method described herein. For example, the inhibition constant of acandidate compound for a mammalian fatty acid desaturase-likepolypeptide can be measured and compared to the inhibition constant fora nematode fatty acid desaturase-like polypeptide.

The aforementioned analyses can be used to identify and/or design amodulator with specificity for nematode fatty acid desaturase-likepolypeptide over vertebrate or other animal (e.g., mammalian) fatty aciddesaturase-like polypeptides. Suitable nematodes to target are anynematodes with the fatty acid desaturase-like proteins or proteins thatcan be targeted by a compound that otherwise inhibits, reduces,activates, or generally affects the activity of nematode fatty aciddesaturase proteins.

Inhibitors of nematode fatty acid desaturase-like proteins can also beused to identify fatty acid desaturase-like proteins in the nematode orother organisms using procedures known in the art, such as affinitychromatography. For example, a specific antibody may be linked to aresin and a nematode extract passed over the resin, allowing any fattyacid desaturase-like proteins that bind the antibody to bind the resin.Subsequent biochemical techniques familiar to those skilled in the artcan be performed to purify and identify bound fatty acid desaturase-likeproteins.

Agricultural Compositions

A compound that is identified as a fatty acid desaturase-likepolypeptide inhibitor can be formulated as a composition that is appliedto plants, soil, or seeds in order to confer nematode resistance. Thecomposition can be prepared in a solution, e.g., an aqueous solution, ata concentration from about 0.005% to 10%, or about 0.01% to 1%, or about0.1% to 0.5% by weight. The solution can include an organic solvent,e.g., glycerol or ethanol. The composition can be formulated with one ormore agriculturally acceptable carriers. Agricultural carriers caninclude: clay, talc, bentonite, diatomaceous earth, kaolin, silica,benzene, xylene, toluene, kerosene, N-methylpyrrolidone, alcohols(methanol, ethanol, isopropanol, n-butanol, ethylene glycol, propyleneglycol, and the like), and ketones (acetone, methylethyl ketone,cyclohexanone, and the like). The formulation can optionally furtherinclude stabilizers, spreading agents, wetting extenders, dispersingagents, sticking agents, disintegrators, and other additives, and can beprepared as a liquid, a water-soluble solid (e.g., tablet, powder orgranule), or a paste.

Prior to application, the solution can be combined with another desiredcomposition such as another anthelmintic agent, germicide, fertilizer,plant growth regulator and the like. The solution may be applied to theplant tissue, for example, by spraying, e.g., with an atomizer, bydrenching, by pasting, or by manual application, e.g., with a sponge.The solution can also be distributed from an airborne source, e.g., anaircraft or other aerial object, e.g., a fixture mounted with anapparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

If the composition is distributed systemically thorough the plant, thecomposition can be applied to tissues other than the leaves, e.g., tothe stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

A skilled artisan would be able to determine an appropriate dosage forformulation of the active ingredient of the composition. For example,the ED50 can be determined as described above from experimental data.The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant or host animal (i.e.non-nematode animal).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding a polypeptide comprising an amino acid sequence that is atleast 95% identical to SEQ ID NO: 10 wherein the polypeptide has fattyacid desaturase activity.
 2. The isolated nucleic acid molecule of claim1 wherein the polypeptide comprises an amino acid sequence that is atleast 98% identical to SEQ ID NO:10.
 3. The isolated nucleic acidmolecule of claim 1 wherein the polypeptide comprises an amino acidsequence that is identical to SEQ ID NO:10.
 4. The isolated nucleic acidmolecule of claim 1 wherein the polypeptide consists of an amino acidsequence that is at least 95% identical to SEQ ID NO:10.
 5. The isolatednucleic acid molecule of claim 4 wherein the polypeptide consists of anamino acid sequence that is at least 98% identical to SEQ ID NO:10. 6.The isolated nucleic acid molecule of claim 4 wherein the polypeptideconsists of an amino acid sequence that is identical to SEQ ID NO:10. 7.A vector comprising the nucleic acid molecule of claim
 1. 8. The vectorof claim 7 wherein the vector is an expression vector.
 9. A recombinantcell harboring the isolated nucleic acid molecule of claim 1.